The induction and repression of benzene and catechol oxidizing capacity of Pseudomonas putida ML2 studied in perturbed chemostat culture

The oxidation of catechol, an intermediate in benzene catabolism, was studied using transient variations in dissolved oxygen tension (DOT) when a succinate limited steady state culture of Pseudomonas putida ML2 was perturbed with a pulse of another substrate. A model was developed and tested for the effect of fluctuations in oxidizing enzyme activity on DOT. It was found that the rate of induction of catechol oxidizing enzymes was independent of dilution rate up to a relative growth rate /_max of 0.75. Only at higher dilution rates was catabolite repression observed.Abbreviations DOT dissolved oxygen tension - K _L a gas transfer coefficient - specific growth rate - _max maximum specific growth rate - K_s substrate saturation constant     

Effect of microbial growth on pore entrance size distribution in sandstone cores

Summary The in situ growth of microorganisms in Berea sandstone cores preferentially plugged the larger pore entrances. The largest pore entrance sizes after microbial plugging ranged from 20 to 38 m, compared with 59 to 69 m before plugging. The pore entrance size distribution of plugged cores was shifted to smaller sizes. A mathematical model based on Poiseuille's equation was found to adequately predict permeability reductions (greater than 90%) caused by microbial growth in the large pore entries.Nomenclature Q volumetric flow rate (L ₃/t) - C orifice constant (dimensionless) - A cross-sectional area (L ₂) - g gravity (L/t ²) - h pieziometric head (L) - _s transmittivity (L ²) - R _e Reynolds number (dimensionless) - a constant (dimensionless) - density (M/L ₃) - viscosity (M/Lt) - d diameter (L) - L length (L) - P pressure change (M/L ₂)     

Toxin-binding properties of cytochrome P450 in Saccharomyces cerevisiae and Kluyveromyces marxianus

Microsomes from Kluyveromyces marxianus GK1005 examined by carbon monoxide difference spectroscopy showed no evidence of cytochrome P450, in contrast to microsomes isolated from a control strain of Saccharomyces cerevisiae. Benzo[a]pyrene produced a typical Type I-binding spectrum with microsomes of both yeasts, with K _s values of 82 M (S. cerevisiae) and 70 M (K. marxianus). While aflatoxin B₁ generated a typical Type I-binding spectrum with microsomes from S. cerevisiae (K _s of 178 M), the toxin did not produce a recognisable binding spectrum with microsomes from K. marxianus.     

L and D presequence peptides derived from the precursor of F1β subunit of the ATP synthase inhibit mitochondrial protein import by interaction with import machinery

We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F₁ subunit of the ATP synthase, pF₁(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F₁ subunit of the ATP synthase, pre-F₁, and the alternative oxidase, pre-AOX, required addition of external ATP, whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b ₂ precursor protein linked to dihydrofolate reductase, pre-b ₂(1, 84)-DHFR was not dependent on ATP. Import of pre-F₁, and pre-AOX was inhibited already at 1 M and 3 M concentration of the L and D enantiomers, whereas inhibition of import of pre-b ₂(1, 84)-DHFR, occurred at concentrations >10 M of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F₁ and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F₁ and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.     

Growth and physiology of potassium-limited chemostat cultures of Paracoccus denitrificans

In potassium-limited chemostat cultures of Paracoccus denitrificans the maximum specific growth rate (µ_max) was found to depend on the input potassium concentration: At 0.21mM µ_max was 0.10–0.11 h^-1; at 0.44 mM 0.15–0.16 h^-1 and at 0.66 mM 0.20–0.21 h^-1. The plots of the specific rates of oxygen-, succinate-and potassium consumption against gave straight lines. The intracellular potassium concentration was a linear function of and varied from 1% (0.13 M) at a value of 0.034 h^-1 to 2.2% (0.29 M) at =0.26 h^-1; the potassium concentration gradient and the potassium concentration in the culture fluid in the steady state were dependent on the input potassium concentration. The potassium concentration gradient varied from 8,900-1,200. At all values 20–25% of the total energy production was used for potassium transport. 350,100 and 30 ATP molecules were calculated to be required to maintain one potassium ion intracellular during 1 h at values of 0.034, 0.197 and 0.257 h^-1 respectively. It is concluded that the amount of circulation of potassium is dependent on the potassium concentration gradient or on the potassium concentration in the culture in the steady state. The dependency of µ_max on the input potassium concentration was explained by the assumption that at low input potassium concentrations the net uptake of potassium (influx-efflux) is not rapidly enough to maintain the high potassium gradient in the existing cells and to establish it in the newly formed cells. At high values and at high input potassium concentrations µ_max is limited by the specific rate of oxygen consumption, which was found to be 11–12 mmol O₂ g dry weight^-1 h^-1 at µ_max for potassium-, succinate-and sulphate-limited chemostat cultures.     

High and low potential states of the chloroplast cytochromeb-559 and thermodynamic control of non-cyclic electron transport

Most of the chloroplastb-559 is high potential at neutral pH as defined by hydroquinone reducibility. FCCP^* (20 M) and antimycin A (50 M) convert high potentialb-559 to a low potential state which can be reduced by ascorbate but not hydroquinone. The low and high potential states of cytochromeb-559 are different forms of the same cytochrome.Three lines of evidence indicate that the cytb-559 oxidized by photosystem I is low potential: (1) theb-559 photooxidized by far-red light in the presence of FCCP (3 M) is low potentialb-559; (2) the amplitude of theb-559 oxidation by far-red light and the amount of low potentialb-559 present in the dark have the same general dependence on pH; (3) inhibitor studies show that plastoquinone mediates the oxidation of cytb-559 by PS I.The well-known stimulation ofb-559 oxidation by far-red light in the presence of FCCP is attributed to FCCP-facilitated photoconversion of high potentialb-559 to a low potential form.It is concluded that if cytb-559 is oxidized by system I light, then it is a low potential form (E _m7+80 mV) which is oxidized. It is not proven, however, that a significant amount of cytb-559 is oxidized by PS I under coupled or physiological conditions.Possible thermodynamic regulation of non-cyclic electron flow involving the distribution between high and low potential forms of cytb-559 is discussed.     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Two types of PS II centres as manifested by light saturation of delayed fluorescence from DCMU-treated chloroplasts

Intensity of 2 s delayed fluorescence (DF) as a function of steady-state actinic light intensity was investigated in pea chloroplasts in the presence of 10 M DCMU. The light saturation curve of DF was approximated by a sum of two hyperbolic components which differ by an order of magnitude in the half-saturating incident light intensity. The relative contribution of the amplitudes of the components was practically independent of cation (Na⁺ and Mg²⁺) concentration and a short-term heating of the chloroplasts at 45°C. The component saturating at low incident light intensity was selectively suppressed by 100 M DCMU or by 1 mol g^-1 Chl oleic acid. DF intensity following excitation by a single saturating 15 s flash was equal to the intensity of the component saturating at a low incident light intensity. Upon flash excitation, the maximum steady-state DF level was found to be attained only after a series of saturating flashes. It is concluded that the two components of the DF light saturation curves are related to PS II centres heterogeneity in quantum yield of stabilization of the reduced primary quinone acceptor.Abbreviations DF Delayed fluorescence - L₁- and L₂-components DF components saturating at low and high incident light intensity, respectively - I incident light intensity - L DF intensity - P₆₈₀ reaction centre chlorophyll of PS II - Q_A and Q_B primary and secondary quinone acceptors of PS II, respectively     

A simple method to estimate percentage parasitism when the host and parasitoid phenologies are unknown: A statistical approach

When host and parasitoid phenologies areunknown the estimation of the generationalpercentage parasitism poses severalmethodological problems. We developed a simplemethod, based on a statistical approach, tosolve that problem. An unbiased estimator wasfound, named _CR, of thegenerational percentage parasitism, and itsperformance was tested through the analysis ofdata from two host-parasitoid systems: Phyllonorycter crataegella (Clemens)(Lepidoptera: Gracillariidae) and Sympiesis marylandensis (Girault)(Hymenoptera: Eulophidae), and Pierisrapae L. (Lepidoptera: Pieridae) and Cotesia (Apanteles) glomerata (L)(Hymenoptera: Braconidae). The _CR estimator proved to be a goodapproximation of the generational percentageparasitism, but its 95% confidence intervalwas very wide. The performance of the _CR estimator was improved and the95% confidence interval was smaller when someinformation on host and parasitoid phenologieswas added. The results suggested that thesmaller interval _CR ± onestandard deviation would contain with a greatprobability the generational percentageparasitism.     

Interrelationships Between Nitrogen Supply and Photosynthetic Parameters in Vicia faba L.

We determined for Vicia faba L the influence of nitrogen uptake and accumulation on the values of photon saturated net photosynthetic rate (P _Nmax), quantum yield efficiency (), intercellular CO₂ concentration (C _i), and carboxylation efficiency (C _e). As leaf nitrogen content (N_L) increased, the converged onto a maximum asymptotic value of 0.0664±0.0049 mol(CO₂) mol(quantum)^–1. Also, as N_L increased the C _i value fell to an asymptotic minimum of 115.80±1.59 mol mol^–1, and C _e converged onto a maximum asymptotic value of 1.645±0.054 mol(CO₂) m^–2 s^–1 Pa^–1 and declined to zero at a N_L-intercept equal to 0.596±0.096 g(N) m^–2. fell to zero for an N_L-intercept of 0.660±0.052 g(N) m^–2. As N_L increased, the value of P _Nmax converged onto a maximum asymptotic value of 33.400±2.563 mol(CO₂) m^–2 s^–1. P _N fell to zero for an N_L-intercept of 0.710±0.035 g(N) m^–2. Under variable daily meteorological conditions the values for N_L, specific leaf area (_L), root mass fraction (R_f), P _Nmax, and remained constant for a given N supply. A monotonic decline in the steady-state value of R_f occurred with increasing N supply. _L increased with increasing N supply or with increasing N_L.     

Changes of photosynthetic parameters in cucumber leaves under Cu, Cd, and Pb stress

The effects of Cu, Cd, and Pb toxicity on photosynthesis in cucumber leaves (Cucumis sativus L.) were studied by the measurements of gas exchange characteristics, chlorophyll (Chl) fluorescence parameters, and Chl content. Concentrations of metals in sequence of 20 M Cu, 20 and 50 M Cd, and 1 000 M Pb decreased the plant dry mass to 50–60 % after 10 d of treatment whereas 50 M of Cu decreased it to 30 %. The content of Cd in leaves of plants treated with 50 M Cd was three times higher than the contents of Cu and Pb after plant treatment with 50 M Cu or 1 000 M Pb. Hence Cd was transported to leaves much better than Cu and Pb. Nevertheless, the net photosynthetic rate and stomatal conductance in leaves treated with 50 M Cu or Cd were similarly reduced. Thus, Cu was more toxic than Cd and Pb for photosynthesis in cucumber leaves. None of the investigated metals decreased internal CO₂ concentrations. Also the effect of metals on potential efficiency of photosystem 2, PS2 (F_v/F_m) was negligible. The metal dependent reduction of PS2 quantum efficiency (_PS2) after plant adaptation in actinic irradiation was more noticeable. This could imply that reduced demand for ATP and NADPH in a dark phase of photosynthesis caused a down-regulation of PS2 photochemistry. Furthermore, in leaves of metal-treated plants the decrease in water percentage as well as lower contents of Chl and Fe were observed. Thus photosynthesis is not the main limiting factor for cucumber growth under Cu, Cd, or Pb stress.This revised version was published online in March 2005 with corrections to the page numbers.     

Transport-dependent alterations of membrane properties of mammalian colon measured using impedance analysis

Summary Direct current (DC) measurement methods have been commonly used to characterize the conductance properties of the mammalian colon. However, these methods provide no information concerning the effects of tissue morphology on the electrophysiological properties of this epithelium. For example, distribution of membrane resistances along narrow fluid-filled spaces such as the lateral intercellular spaces (LIS) or colonic crypts can influence DC measurements of apical and basolateral membrane properties. We used impedance analysis to determine the extent of such distributed resistance effects and to assess the conductance and capacitance properties of the colon. Because capacitance is proportional to membrane area, this method provides new information concerning membrane areas and specific ionic conductances for these membranes.We measured transepithelial impedance under three conditions: (1) control conditions in which the epithelium was opencircuited and bathed on both sides with NaCl–HCO₃ Ringer's solutions, (2) amiloride conditions which were similar to control except that 100 m amiloride was present in the mucosal bathing solution, and (3) mucosal NaCl-free conditions in which mucosal Na and Cl were replaced by potassium and sulfate or gluconate (K⁺ Ringer's). Three morphologically-based equivalent circuit models were used to evaluate the data: (1) a lumped model (which ignores LIS resistance), (2) a LIS distributed model (distributed basolateral membrane impedance) and (3) a crypt-distributed model (distributed apical membrane impedance). To estimate membrane impedances, an independent measurement of paracellular conductance (G _s ) was incorporated in the analysis. Although distributed models yielded improved fits of the data, the distributed and lumped models produced similar estimates of membrane parameters. The predicted effects of distributed resistances on DC microelectrode measurements were largest for the LIS-distributed model. LIS-distributed effects would cause a 12–15% underestimate of membrane resistance ratio (R _a /R _b ) for the control and amiloride conditions and a 34% underestimate for the K Ringer's condition. Distributed resistance effects arising from the crypts would produce a 1–2% overestimate ofR _a /R _b .Apical and basolateral membrane impedances differed in the three different experimental conditions. For control conditions, apical membrane capacitance averaged 21 F/cm² and the mean apical membrane specific conductance (G _a-norm) was 0.17 mS/F. The average basolateral membrane capacitance was 11 F/cm² with a mean specific conductance (G _b-norm) of 1.27 mS/F.G _a-norm was decreased by amiloride or K⁺ ringer's to 0.07 mS/gmF and 0.06 mS/F, respectively. Basolateral conductance was also reduced by amiloride, whereas capacitance was unchanged (G _b-norm=0.97 mS/F). For the K⁺ Ringer's condition, both basolateral conductance and capacitance were greatly increased such thatG _b-norm was not significantly different from the control condition.     

Effects of random motility on growth of bacterial populations

A spatially distributed mathematical model is developed to elucidate the effects of chemical diffusion and cell motility as well as cell growth, death, and substrate uptake on steady-state bacterial population growth in a finite, one-dimensional, nonmixed region. The situation considered is growth limited by a diffusing substrate from an adjacent phase not accessible to the bacteria. Chemotactic movement is not considered in this paper; we consider only randomwalk-type random motility behavior here. The following important general concepts are suggested by the results of our theoretical analysis: (a) The significance of random motility effects depends on the magnitude of the ratio/kL ², where is the bacterial random motility coefficient,k is the growth rate constant, andL is the linear dimension of the confined growth region. (b) In steady-state growth in a confined region, the bacterial population size decreases as increases. (c) The effect of on population size can be great; in fact, sometimes relative population sizes of two species can be governed primarily by the relative values of rather than by the relative values ofk.     

Transport of inorganic phosphate inPseudomonas aeruginosa

Inorganic phosphate transport by wild-typePseudomonas aeruginosa cells grown in a phosphate-limited medium involves a biphasic process. The uptake obeys Michaelis-Menten kinetics with respective apparentK _m values of 1.1 M and 10 M for the high- and low-affinity systems. These systems may be also differentiated by their sensitivity to osmotic shock, by their specificity towards phosphite, pyrophosphate, arsenate, and some phosphonates and also by their energy requirements. The two phosphate transport systems fromP. aeruginosa are compared with the two major systems (Pst and Pit) characterized inEscherichia coli.     

Leaf photosynthetic characteristics of seedlings of actinorhizal Alnus spp. and Elaeagnus spp.

Single leaf photosynthetic characteristics of Alnus glutinosa, A. incana, A. rubra, Elaeagnus angustifolia, and E. umbellata seedlings conditioned to ambient sunlight in a glasshouse were assessed. Light saturation occurred between 930 and 1400 mol m^-2s^-1 PAR for all species. Maximum rates of net photosynthesis (Pn) measured at 25°C ranged from 12.8 to 17.3 mol CO₂m^-2s^-1 and rates of dark respiration ranged from 0.74 to 0.95 mol CO₂m^-2s^-1. These values of leaf photosynthetic variables are typical of early to midsuccessional species. The rate of Pn measured at optimal temperature (20°C) and 530mol m^-2s^-1 PAR was significantly (p<0.01) correlated with leaf nitrogen concentration (r=0.69) and negatively correlated with the mean area of a leaf (r=–0.64). We suggest that the high leaf nitrogen concentration and rate of Pn observed for Elaeagnus umbellata and to a lesser degree for E. angustifolia are genetic adaptations related to their crown architecture.Abbreviations Pn net photosynthesis     

Heterogeneous Long Chain Acyl-CoA Synthetases Control Distribution of Individual Fatty Acids in Newly-Formed Glycerolipids of Neuronal Cells Undergoing Neurite Outgrowth

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [³H]glycerol, [³H]oleic acid (OA) or [³H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 M), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [³H]OA into glycerolipids by 30–90%, and increased by about 60% the accumulation of free [³H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [³H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 M, respectively; and in cytoplasm, 6 and 0.6 nmol/ min/mg protein and 38 and 60 M, respectively. Triacsin C (2–33 M) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC₅₀ and apparent Ki values were 13.5 M and 14 M in microsomes, and 3.8 M and 4 M in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.     

Effect of Naloxone on the Activity of Cl–-Activated Mg2+-ATPase from Plasma Membrane Fraction of Bream Brain (Abramis brama L.) in the Presence of GABAa-ergic Substances

We studied the effect of naloxone—an antagonist of the opioid receptors—on sensitivity of Cl^–-activated Mg²⁺-ATPase from the plasma membrane fraction of bream brain (Abramis brama L.) to GABA_a-ergic substances. Preincubation of the plasma membranes with 1–100 M naloxone increased the basal Mg²⁺-ATPase activity and suppressed its activation by chloride ions. The same effects were observed in the presence of the agonists of GABA_a/benzodiazepine receptors: 0.1–100 M GABA, 1–500 M pentobarbital, and 0.1–100 M phenazepam. Naloxone (10 M) inhibited activation of the basal Mg²⁺-ATPase by the studied ligands and restored the enzyme sensitivity to Cl^–. However, the effect of naloxone was not observed in the presence of high concentrations of pentobarbital (500 M) and phenazepam (100 M). The obtained data show that naloxone modulates the activity of Cl^–-activated Mg²⁺-ATPase from the plasma membranes of bream brain and antagonizes the GABA_a receptor ligands.