Xylem sap chemistry: seasonal changes in timberline conifers <Emphasis Type="Italic">Pinus cembra</Emphasis>, <Emphasis Type="Italic">Picea abies</Emphasis>, and <Emphasis Type="Italic">Larix decidua</Emphasis>

The seasonal course of xylem sap parameters (electrical conductivity EC, potassium concentration [K⁺], and pH) of three conifers (Pinus cembra, Picea abies, and Larix decidua) growing at the alpine timberline was monitored. We also looked into possible effects of [K⁺] and pH on the difference in hydraulic conductivity (Δk_s). In all studied species, EC, [K⁺], and pH varied considerably over the year, with pH ranging between 7.3 (February) and 5.8 (June) and [K⁺] changing between 0.4 (January) and 2.5 mM (June). The Δk_s was overall low with positive values during winter (up to +20 %) and negative values in summer (-15 % in August). Samples perfused with alkaline solutions showed higher Δk_s. Xylem sap parameters in all conifers under study were surprisingly variable over the year thus indicating either effects upon seasonal changes in environmental factors or active adjustments, or both. Although Δk_s values over the year were minor, observed induction of Δk_s by high pH might indicate a role for hydraulic adjustment in harsh winter periods.     

New relations for steady-state enzyme kinetics and their application to rapid equilibrium assumption

With the use of graph theory, new relations for steady-state enzyme kinetics are derived and strictly proved for an arbitrary mechanism of enzyme-catalyzed reaction containing a reversible segment. Using these relations, a general principle for rapid equilibrium assumption is formulated and proved: the reversible bound segment can be considered as an equilibrium segment only when the values of the base trees that are not proper to this segment can be neglected (within prescribed accuracy) in relation to the values of the base trees that belong to this segment. In contrast with the foreign base trees, the base trees that are proper to the segment have the following properties: the tree that is directed to the base within this segment does not contain edges leaving this segment; and the tree that is directed to the base outside the segment contains only one edge leaving this segment. Equilibrium variations are assessed for steady-state concentrations of intermediates in the equilibrium segment, numerical expressions are obtained for the accuracy of determination of intermediate concentrations as well as for the accuracy of determination of the rate of enzyme-catalyzed reaction under rapid equilibrium assumption.     

Kinetics of isotope exchange at equilibrium for a one substrate-one product enzyme mechanism

It is shown that the general solution of Morales, Horovitz &; Botts (1962) describing isotope exchange kinetics at equilibrium for the one substrateone product enzyme mechanism:

$\text{E+S?X?E+P}$

reduces to the steady-state solution normally applied to experimental data, under usual experimental conditions.The analysis provides a firmer theoretical basis for the equations generally used in analysing kinetic data from isotope exchange experiments.     

Theoretical Aspects of Differential Scanning Calorimetry as a Tool for the Studies of Equilibrium Thermodynamics in Pharmaceutical Solid Phase Transitions

Although differential scanning calorimetry (DSC) is a non-equilibrium technique, it has been used to gain energetic information that involves phase equilibria. DSC has been widely used to characterize the equilibrium melting parameters of small organic pharmaceutical compounds. An understanding of how DSC measures an equilibrium event could make for a better interpretation of the results. The aim of this mini-review was to provide a theoretical insight into the DSC measurement to obtain the equilibrium thermodynamics of a phase transition especially the melting process. It was demonstrated that the heat quantity obtained from the DSC thermogram (ΔH) was related to the thermodynamic enthalpy of the phase transition (ΔH ^P ) via: ΔH?=?ΔH ^P /(1?+?K ^??1) where K was the equilibrium constant. In melting, the solid and liquefied phases presumably coexist resulting in a null Gibbs free energy that produces an infinitely larger K. Thus, ΔH could be interpreted as ΔH ^P. Issues of DSC investigations on melting behavior of crystalline solids including polymorphism, degradation impurity due to heating in situ, and eutectic melting were discussed. In addition, DSC has been a tool for determination of the impurity based on an ideal solution of the melt that is one of the official methods used to establish the reference standard.     

Measurements of the plasma density in the FTU tokamak by a pulsed time-of-flight X-wave refractometer

On-line control over the plasma density in tokamaks (especially, in long-term discharges) requires reliable measurements of the averaged plasma density. For this purpose, a new method of density measurements—a pulsed time-of-flight plasma refractometry—was developed and tested in the T-11M tokamak. This method allows one to determine the averaged density from the measured time delay of nanosecond microwave pulses propagating through the plasma. For an O-wave, the measured time delay is proportional to the line-averaged density and is independent of the density profile (f?f _p ) τ_o ≈ k _o \(\tfrac{1}{{f^2 }}\mathop \smallint \limits_l \) N(x dx. A similar formula is valid for an X-wave: τ_X = ≈ k _x \(\tfrac{{f^2 + f_c^2 }}{{(f^2 - f_c^2 )^2 }}\mathop \smallint \limits_l \) N(x)dx. Here, f is the frequency of the probing wave, f _p is the plasma frequency, l= 4 a is the path length for two-pass probing in the equatorial plane, a is the plasma minor radius, k _O and k _X are numerical factors, f _c is the electron-cyclotron frequency at the axis of the plasma column, and f _p ?f _c , f. Measurements of the time delay provide the same information as plasma interferometry, though they do no employ the effect of interference. When the conditions f _p ?f _c , f are not satisfied, the measured time delay depends on the shape of the density profile. In this case, in order to determine the average density regardless of the density profile, it is necessary to perform simultaneous measurements at several probing frequencies in order to determine the average density. In ITER (Bt ～ 5T), a spectral window between the lower and upper cutoff frequencies in the range of 50–100 GHz can be used for pulsed time-of-flight X-wave refractometry. This appreciably simplifies the diagnostics and eliminates the problem of the first mirror. In this paper, the first results obtained in the FTU tokamak with a prototype of the ITER pulsed time-of-flight refractometer are presented. The geometry and layout of experiments similar to the planned ITER experiments are described. The density measured by pulsed time-of-flight refractometry is shown to agree well with the results obtained in FTU with a two-frequency scanning IR interferometer. The results obtained are analyzed, and the future experiments are discussed.     

Cloning and Functional Analysis of <Emphasis Type="Italic">SaCLCc1</Emphasis>, a Gene Belonging to the Chloride Channel Family (<Emphasis Type="Italic">CLC</Emphasis>), from the Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe²⁺-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn²⁺. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl^– ions.     

A generalized theory of particulate electron conduction enzymes applied to cytochrome oxidase. A theory of coupled electron and/or ion transport applied to pyruvate carboxylase

A previously developed theory of particulate electron conduction enzymes was based on a model of an enzyme particle catalyzing the oxidation-reduction of two different substrates at two different enzymatic sites on the same particle with conduction of electrons between the two sites through the enzyme particle. Using the simplifying assumption that the percent reduction of the second substrate is held constant, there was previously shown to be a hyperbolic relationship between the first order rate constant (k′) and the sum (C _x ) of oxidized plus reduced substrate, of the formk′=α/(C _x +β), where α and β are positive constants. It is shown here that if this simplifying assumption is omitted, a positive constant is added to the right hand side of this equation, which describes exactly the experimental data of Smith and conrad on cytochrome oxidase. If electron transport is assumed to be coupled to ion transport, this equation becomesk′=(α/C _x )?γ (where γ is a positive constant) which describes the experimental data of Eadie and Gale on pyruvic carboxylase of yeast. It seems probable that the same theory is applicable to coupled ion-ion transport and coupled electron-electron transport in both membranous systems, and in particulate preparations consisting of membrane fragments.     

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Background

Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure.

Results

Transfection conditions were first optimized to achieve expression levels between 10 and 18?mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4–9?mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K_d) of 5?nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k_cat (7.2?±?0.5?min^??1) and K_M (1.2?±?0.3?μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption.

Conclusions

The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k_cat and K_M values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

     

Purification and Characterization of a Novel (<Emphasis Type="Italic">R</Emphasis>)-1-Phenylethanol Dehydrogenase from <Emphasis Type="Italic">Lysinibacillus</Emphasis> sp. NUST506

A novel (R)-1-phenylethanol dehydrogenase was successfully purified from Lysinibacillus sp. NUST506 by preparative polyacrylamide gel electrophoresis. The enzyme is a NAD+-dependent oxidoreductase. The molecular weight of the (R)-1-phenylethanol dehydrogenase measured by SDS-PAGE was about 28 kDa. Furthermore, the optimal reaction conditions for the oxidative reaction were 70°C and pH 9.5 and for the reductive reaction were 65°C and pH 6.5. Under the optimal conditions, the K_M and k_cat values with (R)-1-phenylethanol as a substrate were found to be 0.78 mM and 123 s^–1 and with acetophenone they were 0.56 mM and 125 s^–1, respectively. The (R)-1-phenylethanol dehydrogenase became more stable at pH 9.5 in comparison with pH 5.0 and high stability was noticed at 4 and 37°C. Properties of the enzyme place it as a promising candidate for industrial applications.     

Influence of a Nitrogen Admixture on the Anomalous Memory Effect in the Breakdown of Low-Pressure Argon in a Long Discharge Tube

The memory effect (the dependence of the dynamic breakdown voltage U _b on the time interval τ between voltage pulses) in pulse-periodic discharges in pure argon and the Ar + 1%N₂ mixture was studied experimentally. The discharge was ignited in a 2.8-cm-diameter tube with an interelectrode distance of 75 cm. The measurements were performed at gas pressures of P = 1, 2, and 5 Torr and discharge currents in a steady stage of the discharge of I = 20 and 56 mA. Breakdown was produced by applying positive-polarity voltage pulses, the time interval between pulses being in the range of τ = 0.5–40 ms. In this range of τ values, a local maximum (the anomalous memory effect) was observed in the dependence U _b (τ). It is shown that addition of nitrogen to argon substantially narrows the range of τ values at which this effect takes place. To analyze the measurement results, the plasma parameters in a steady-state discharge (in both pure argon and the Ar + 1%N₂ mixture) and its afterglow were calculated for the given experimental conditions. Analysis of the experimental data shows that the influence of the nitrogen admixture on the shape of the dependence U _b (τ) is, to a large extent, caused by the change in the decay rate of the argon afterglow plasma in the presence of a nitrogen admixture.     

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium <Emphasis Type="Italic">Alteromonas</Emphasis> sp. L82

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni²⁺ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a K_m of 0.55 mM, a V_max of 83.6 U/mg, a k_cat of 74.3 s^-1 and k_cat/K_m of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.     

Composition of the genera <Emphasis Type="Italic">Gnaptorina</Emphasis> Reitter and <Emphasis Type="Italic">Pseudognaptorina</Emphasis> Kaszab of the tribe Blaptini (Coleoptera,Tenebrionidae)

Based on the structure of the segments of the male fore and middle tarsi, three subgenera are distinguished in the genus Gnaptorina Reitter: Gnaptorina s. str. (brushes or tufts of pale setae are present on the sole surface of the 1st–3rd segments of the fore tarsus and also on the 1st and 2nd segments of the middle tarsus, the type species Gnaptorina felicitana Reitter, 1887), Boreoptorina subgen. n. (a brush is present on the sole surface of the 1st segment of the fore tarsus, but is absent on the sole surface of the segments of middle tarsus, the inner margin of the male fore tibia with a hairy brush, the type species Gnaptorina cordicollis G. Medvedev, 1998), and Hesperoptorina subgen. n. (a flat hairy brush is present only on the sole surface of the 1st segment of the fore tarsus, type species Gnaptorina brucei Blair, 1923). The genus Pseudognaptorina Kaszab, 1977 is considered monotypical. Lectotypes of four species of Gnaptorina are designated.     

Cloning,purification and characterization of a cellulase-free xylanase from <Emphasis Type="Italic">Geobacillus thermodenitrificans</Emphasis> AK53

Geobacillus thermodenitrificans AK53 xyl gene encoding xylanase was isolated, cloned and expressed in Escherichia coli. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, biochemical properties of the enzyme were determined. The kinetic studies for GthAK53Xyl showed K_M value to be 4.34 mg/mL (for D-xylose) and V_max value to be 2028.9 μmoles mg^–1 min^–1. The optimal temperature and pH for enzyme activity were found out to be 70°C and 5.0, respectively. The expressed protein showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644). Metal cations Mg²⁺ and Mn²⁺ were found to be required for the enzyme activity, however, Co²⁺, Hg²⁺, Fe²⁺ and Cu²⁺ ions caused inhibitor effect on it. GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo-fashion. The action of the enzyme on xylan from oat spelt produced xylobiose and xylopentose. The reported results are suggestive of a xylanase exhibiting desirable kinetics, stability parameters and metal resistance required for the efficient production of xylobiose at industrial scale.     

Mining statistically-solid <Emphasis Type="Italic">k</Emphasis>-mers for accurate NGS error correction

Background

NGS data contains many machine-induced errors. The most advanced methods for the error correction heavily depend on the selection of solid k-mers. A solid k-mer is a k-mer frequently occurring in NGS reads. The other k-mers are called weak k-mers. A solid k-mer does not likely contain errors, while a weak k-mer most likely contains errors. An intensively investigated problem is to find a good frequency cutoff f₀ to balance the numbers of solid and weak k-mers. Once the cutoff is determined, a more challenging but less-studied problem is to: (i) remove a small subset of solid k-mers that are likely to contain errors, and (ii) add a small subset of weak k-mers, that are likely to contain no errors, into the remaining set of solid k-mers. Identification of these two subsets of k-mers can improve the correction performance.

Results

We propose to use a Gamma distribution to model the frequencies of erroneous k-mers and a mixture of Gaussian distributions to model correct k-mers, and combine them to determine f₀. To identify the two special subsets of k-mers, we use the z-score of k-mers which measures the number of standard deviations a k-mer’s frequency is from the mean. Then these statistically-solid k-mers are used to construct a Bloom filter for error correction. Our method is markedly superior to the state-of-art methods, tested on both real and synthetic NGS data sets.

Conclusion

The z-score is adequate to distinguish solid k-mers from weak k-mers, particularly useful for pinpointing out solid k-mers having very low frequency. Applying z-score on k-mer can markedly improve the error correction accuracy.

     

Purification and Characterization of Exo-Inulinase from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. d9 Strain

This study intended to purify and characterise exo-inulinase of diesel-degrading Paenibacillus sp. D9. The whole genome sequencing of Paenibacillus sp. D9 revealed to possess the sacC gene that is encoded as exo-inulinase/levanase. This isolate was capable of producing a maximum of 50.9 IU/mL of exo-inulinase activity within 3 days at 30?°C, 200 rpm and pH of 7.0 on minimal salt medium agar supplemented with 1% (w/v) inulin. An exo-inulinase of 58.5 kDa was purified using ammonium sulphate precipitation, HiTrap QFF column and MMC column chromatographies with a specific activity of 4333 IU/mg, 7.1% recovery and a 4.3-fold increase in purity. The purified D9 exo-inulinase had temperature and pH optimum at 40?°C and pH 4.0, respectively, with the Michaelis constant of 5.5 mM and a maximal velocity of 476.2 IU/mg, respectively. Catalytic constant, k _cat was calculated to be 42.6 s^?1 with a catalytic efficiency (k _cat /K _m ) of 7.6 s^?1 mM^?1. The presence of Ca²⁺ enhanced the activity of D9 exo-inulinase while Hg²⁺ completely inhibited the activity, other compounds such as Fe³⁺ and Cu²⁺ had an inhibitory effect. The results of amino acid alignment and the complete degradation of inulin into fructose by the purified enzyme confirmed that inulinase from Paenibacillus sp. D9 is an exo-form. The phylogenetic tree based on the protein sequences indicates that bacterial exo-inulinases possess a common ancestry.     

Drift stabilization of internal resistive-wall modes in tokamaks

The problem of drift stabilization of the internal resistive-wall modes (RWMs) in tokamaks is theoretically investigated. The basic assumption of the model is that, when the drift effects are neglected, these modes are unstable in the absence of a conducting wall and stable in the presence of a close-fitting perfectly conducting wall. In the former case, the instability condition is expressed as Δ′_∞>0, where Δ′_∞ is the matching parameter calculated under the assumption that the wall is removed to infinity. In the latter case, one has Δ _W ^′ <0, where Δ _W ^′ is the external matching parameter of tearing modes calculated assuming a perfectly conducting wall at the plasma boundary. In the case with a resistive wall, the relevant parameter can be either Δ′_∞ or Δ _W ^′ , depending on whether the value of the dimensionless parameter ωτ_s/2m is small or large, respectively (here ω is the mode frequency, τ_s is the resistive time constant of the wall, and m is the poloidal mode number). In the presence of drift effects, the mode frequency ω is approximately equal to the electron drift frequency, ω≈ω_*e. The value of the parameter ω_*eτ_s/2m, which therefore determines the behavior of internal RWMs, is estimated for several existing tokamaks, namely, AUG (ASDEX-Upgrade), DIII-D, JET, TFTR, and JT-60U, as well as for the projected ITER-FEAT. It is shown that, although drift effects do not stabilize internal RWMs in current devices, they should be efficient in suppressing these modes in reactor-grade tokamaks.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

The invariance of production per unit of food consumed in fish populations

The amount of biomass production per unit of food consumed (P/Q) represents an important quantity in ecosystem functioning, because it indicates how efficient a population transforms ingested food into biomass. Several investigations have noticed that P/Q remains relatively constant (or invariant) across fish population that feed at the same food-type level (carnivorous/herbivorous). Nevertheless, theoretical explanation for this invariant is still lacking. In this paper, we demonstrate that P/Q remains invariant across fish populations with stable-age distribution. Three key assumptions underpin the P/Q invariant: (1) the ratio between natural mortality M and von Bertalanffy growth parameter k (M/k ratio) should remain invariant across fish populations; (2) a parameter defining the fraction of ingested food available for growth needs to remain constant across fish that feed at the same trophic level; (3) third, the ratio between length at age 0 (\(l_0\)) and asymptotic length (\(l_\infty\)) should be constant across fish populations. The influence of these assumptions on the P/Q estimates were numerically assessed considering fish populations of different lifespan. Numerical evaluations show that the most critical condition highly relates to the first assumption, M/k. Results are discussed in the context of the reliability of the required assumption to consider the P/Q invariant in stable-age distributed fish populations.