黄土高原冬小麦地N2O排放

从2007年7月1日到2009年6月30日对黄土高原冬小麦地氧化亚氮(N₂O)排放采用静态箱气相色谱法进行了为期2a 的监测。设置2个处理,有小麦田(有小麦生长),无小麦田(出芽初期拔去麦苗)。研究结果表明有小麦田、无小麦田N₂O排放量年际变化不大。有小麦田年均的N₂O 排放量为2.05 kg · N₂O · hm^-2 · a^-1,无小麦田年均的N₂O 排放量为2.28 kg · N₂O · hm^-2 · a^-1 。在冻融交替期,施肥后、翻地后和降雨后无小麦田和有小麦田N₂O排放明显增加,N₂O的季节变化受到这些短期事件的显著影响;有小麦田N₂O排放与地温(P<0.01),气温(P<0.01)和WFPS(P<0.05)显著相关,而无小麦田N₂O排放与这些环境土壤因子都不相关;有小麦田和无小麦田两个处理土壤的WFPS通常都低于60%,可以推断在本地区,硝化反应是N₂O的重要生成源。     

La and Gd induce shape change of giant unilamellar vesicles of phosphatidylcholine

Lanthanides such as La³⁺ and Gd³⁺ are well known to have large effects on the function of membrane proteins such as mechanosensitive ionic channels and voltage-gated sodium channels, and also on the structure of phospholipid membranes. In this report, we have investigated effects of La³⁺ and Gd³⁺ on the shape of giant unilamellar vesicle (GUV) of dioleoylphosphatidylcholine (DOPC-GUV) and GUV of DOPC/cholesterol by the phase-contrast microscopy. The addition of 10-100 μM La³⁺ (or Gd³⁺) through a 10-μm diameter micropipette near the DOPC-GUV (or DOPC/cholesterol-GUV) triggered several kinds of shape changes. We have found that a very low concentration (10 μM) of La³⁺ (or Gd³⁺) induced a shape change of GUV such as the discocyte via stomatocyte to inside budded shape transformation, the two-spheres connected by a neck to prolate transformation, and the pearl on a string to cylinder (or tube) transformation. To understand the effect of these lanthanides on the shape of the GUV, we have also investigated phase transitions of 30 μM dipalmitoylphosphatidylcholine-multilamellar vesicle (DPPC-MLV) by the ultra-sensitive differential scanning calorimetry (DSC). The chain-melting phase transition temperature and the L_β′ to P_β′ phase transition temperature of DPPC-MLV increased with an increase in La³⁺ concentration. This result indicates that the lateral compression pressure of the membrane increases with an increase in La³⁺ concentration. Thereby, the interaction of La³⁺ (or Gd³⁺) on the external monolayer membrane of the GUV induces a decrease in its area (A^ex), whereas the area of the internal monolayer membrane (Aⁱⁿ) keeps constant. Therefore, the shape changes of the GUV induced by these lanthanides can be explained reasonably by the decrease in the area difference between two monolayers (ΔA=A^ex−Aⁱⁿ).     

Sequential degradation of <Emphasis Type="Italic">p</Emphasis>-cresol by photochemical and biological methods

Sequential photo-and biodegradation of p-cresol was studied using a mercury lamp, as well as KrCl and XeCl excilamps. Preirradiation of p-cresol at a concentration of 10^?4 M did not affect the rate of its subsequent biodegradation. An increase in the concentration of p-cresol to 10^?3 M and in the duration preliminary UV irradiation inhibited subsequent biodegradation. Biodegradation of p-cresol was accompanied by the formation of a product with a fluorescence maximum at 365 nm (λ_ex = 280 nm), and photodegradation yielded a compound fluorescing at 400 nm (λ_ex = 330 nm). Sequential UV and biodegradation led to the appearance of bands in the fluorescence spectra that were ascribed to p-cresol and its photolysis products. It was shown that sequential use of biological and photochemical degradation results in degradation of not only the initial toxicant but also the metabolites formed during its biodegradation.     

Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na⁺]_in) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H⁺) or sodium ions (Na⁺), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H⁺- and Na⁺-driven components that functions as a sodium motor. Changing external sodium concentration ([Na⁺]_ex) in the range 1–85 mM resulted in changes in [Na⁺]_in between 5–14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na⁺]_in and [Na⁺]_ex, and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2–3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.     

Syntheses, structures, and photoluminescent properties of four d metal-quinolinato coordination polymers with similar rod-like SBUs

Four M^II quinolinato complexes, [Zn₂(quin)₂(H₂O)₃]_n (1), [Zn(quin)(H₂O)₂]_n (2), [Zn(quin)(H₂O)]_n (3) and [Cd(quin)]_n (4) (H₂quin = 2,3-pyridinedicarboxylic acid or quinolinic acid), have been hydrothermally synthesized and structurally characterized. X-ray diffraction analyses reveal that all of these four complexes are constructed from similar rod-like SBUs, [M(quin)]_n (M = Zn or Cd). Complexes 1 and 2 have similar 1-D box-like chains but different packing structures; complex 3 has a 2-D grid-like network and complex 4 has an unusual 2-D bilayer structure. Due to the different structural features, these complexes exhibit different photoluminescent emissions: complex 1 at 439 nm (λ_ex = 345 nm), complex 2 at 428 nm (λ_ex = 360 nm), complex 3 at 508 nm (λ_ex = 304 nm) and complex 4 at 500 nm (λ_ex = 324 nm).     

Hydrogenase activity and proton-motive force generation by Escherichia coli during glycerol fermentation

Proton motive force (Δp) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical—the membrane potential (?φ) and chemical—the pH transmembrane gradient (ΔpH), were established and the effects of external pH (pH_ex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pH_ex at pH 7.5 and 6.5, respectively; and it was higher than pH_ex at pH 5.5. At high pH_ex, the increase of ?φ (?130 mV) was only partially compensated by a reversed ΔpH, resulting in a low Δp. At low pH_ex ?φ and consequently Δp were decreased. The generation of Δp during glycerol fermentation was compared with glucose fermentation, and the difference in Δp might be due to distinguished mechanisms for H⁺ transport through the membrane, especially to hydrogenase (Hyd) enzymes besides the F₀F₁-ATPase. H⁺ efflux was determined to depend on pH_ex; overall and N,N’-dicyclohexylcarbodiimide (DCCD)-inhibitory H⁺ efflux was maximal at pH 6.5. Moreover, ΔpH was changed at pH 6.5 and Δp was different at pH 6.5 and 5.5 with the hypF mutant lacking all Hyd enzymes. DCCD-inhibited ATPase activity of membrane vesicles was maximal at pH 7.5 and decreased with the hypF mutant. Thus, Δp generation by E. coli during glycerol fermentation is different than that during glucose fermentation. Δp is dependent on pH_ex, and a role of Hyd enzymes in its generation is suggested.     

Ryanodine receptor 1 mediates Ca2+ transport and influences the biomechanical properties in RBCs

Ryanodine receptors (RyRs) are a family of Ca²⁺ channel proteins that mediate the massive release of Ca²⁺ from the endoplasmic reticulum into the cytoplasma. In the present study, we manipulated the incorporation of RyR1 into RBC membrane and investigated its influences on the intracellular Ca²⁺ ([Ca²⁺]_in) level and the biomechanical properties in RBCs. The incorporation of RyR1 into RBC membranes was demonstrated by both immunofluorescent staining and the change of [Ca²⁺]_in of RBCs. In the presence of RyR1, [Ca²⁺]_in showed biphasic changes, i.e., it increased with the extracellular Ca²⁺ ([Ca²⁺]_ex) up to 5 μM and then decreased with the further increase of [Ca²⁺]_ex. However, [Ca²⁺]_in remained constant in the absence of the RyR1. The results of biomechanical measurements on RBCs, including deformability, osmotic fragility, and membrane microviscosity, reflected similar biphasic changes of [Ca²⁺]_in mediated by RyR1 with the increases of [Ca²⁺]_ex. Therefore, it is believed that RyR1 can incorporate into RBC membrane in vitro, and mediate Ca²⁺ influx, and then regulate RBC biomechanical properties. This information suggests that RBCs may serve as a model to study the function of RyR1 as a Ca²⁺ release channel.     

Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter

Multiscale reactive molecular dynamics simulations are used to study proton transport through the central region of ClC-ec1, a widely studied ClC transporter that enables the stoichiometric exchange of 2 Cl^– ions for 1 proton (H⁺). It has long been known that both Cl^– and proton transport occur through partially congruent pathways, and that their exchange is strictly coupled. However, the nature of this coupling and the mechanism of antiporting remain topics of debate. Here multiscale simulations have been used to characterize proton transport between E203 (Glu_in) and E148 (Glu_ex), the internal and external intermediate proton binding sites, respectively. Free energy profiles are presented, explicitly accounting for the binding of Cl^– along the central pathway, the dynamically coupled hydration changes of the central region, and conformational changes of Glu_in and Glu_ex. We find that proton transport between Glu_in and Glu_ex is possible in both the presence and absence of Cl^– in the central binding site, although it is facilitated by the anion presence. These results support the notion that the requisite coupling between Cl^– and proton transport occurs elsewhere (e.g., during proton uptake or release). In addition, proton transport is explored in the E203K mutant, which maintains proton permeation despite the substitution of a basic residue for Glu_in. This collection of calculations provides for the first time, to our knowledge, a detailed picture of the proton transport mechanism in the central region of ClC-ec1 at a molecular level.     

Simultaneous determination of the lactone and carboxylate forms of the camptothecin derivative CPT-11 and its metabolite SN-38 in plasma by high-performance liquid chromatography

CPT-11 (irinotecan) and mainly its metabolite SN-38 are potent antitumor derivatives of camptothecin. As the active lactone forms of both CPT-11 and SN-38 exist in pH-dependent equilibrium with their respective less potent open-ring hydroxy acid species, the simultaneous monitoring of both forms of both compounds is relevant. CPT-11 and SN-38 derivatives have quite different fluorescence responses. In order to avoid any compromise on the wavelength setting, we developed chromatographic conditions allowing simple automated wavelength setting changes which have been prevented using existing methods involving conventional C₁₈ columns. This was achieved by means of a Symmetry C₁₈ column combined to a gradient elution program using acetonitrile and 75 mM ammonium acetate plus 7.5 mM tetrabutylammonium bromide at pH 6.4. The developed conditions allowed an elution order suitable for a simple automated wavelength change in respect to reliable peak integration. CPT-11 and SN-38 derivatives were detected at λ_ex=362 nm/λ_em=425 nm and λ_ex=375 nm/λ_em=560 nm, respectively. The developed method allowed the detection of amounts less than 3 pg of each derivative injected on column. The method was successfully applied to pharmacokinetic and toxicokinetic studies in rat and dog.     

Surprises from an Unusual CLC Homolog

The chloride channel (CLC) family is distinctive in that some members are Cl⁻ ion channels and others are Cl⁻/H⁺ antiporters. The molecular mechanism that couples H⁺ and Cl⁻ transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl⁻ and H⁺ transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl⁻-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl⁻ transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO₄²⁻ strongly competes with Cl⁻ uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl⁻/H⁺ antiporter, even though it contains an isoleucine at the Glu_in position that was previously thought to be a critical part of the H⁺ pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu_in site in CLC-ck2 does not increase H⁺ flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu_ex) in CLC-ck2 prevents H⁺ flux. Hence, Glu_ex, but not Glu_in, is critical for H⁺ permeation in CLC proteins.The chloride channel (CLC) family includes both Cl⁻ ion channels and Cl⁻/H⁺ antiporters (1). The ion channels allow Cl⁻ to diffuse passively down an electrochemical gradient, and antiporters couple the movement of chloride and protons in opposite directions across cellular membranes. So far, the only known CLC structures are those of antiporters (2–4). On the basis of sequence similarity and functional studies, it is thought that the basic structures of the ion channels and antiporters are similar, and that slight structural differences account for these diverse functions. Understanding how the CLC family has evolved to allow proteins of similar structure to carry out two distinct mechanisms remains a critical goal.In the Escherichia coli antiporter CLC-ec1, two glutamates, Glu_ex (E148) and Glu_in (E203), are absolutely required for H⁺ transport (5,6). Glu_ex is conserved in both CLC ion channels and antiporters. Glu_in is conserved only in antiporters and is instead a hydrophobic valine in all of the known ion channels. Hence, it was proposed that both Glu_in and Glu_ex are necessary to transfer protons through CLC antiporters (6). Studies of the CLC-4 and CLC-5 antiporters supported the notion that Glu_in and Glu_ex play critical roles in H⁺ transport (7,8). Surprisingly, however, recent experiments revealed that although the red algae homolog CmCLC contains a threonine at the Glu_in position, it is still Cl⁻/H⁺ antiporter (3). It is unknown whether this threonine has a shifted pKa that allows it to transfer protons or whether the H⁺ transport in CmCLC does not require a protonatable residue at this position. Further blurring the role of Glu_in, the CLC-0 ion channel, which contains a valine at the Glu_in position, requires slow transmembrane H⁺ transport for channel gating (9).To probe the molecular requirements for Cl⁻ and H⁺ transport in CLC proteins, we characterized a novel homolog from Citrobacter koseri called CLC-ck2. CLC-ck2 is 21% identical and 37% similar in amino acid sequence to CLC-ec1. CLC-ck2 contains an isoleucine at the Glu_in position, and hence we originally hypothesized that this protein would act as an ion channel. Additionally, CLC-ck2 lacks several amino acids that coordinate the central and internal Cl⁻-binding sites in CLC-ec1, most notably the GSGIP motif (Fig. S1 in the Supporting Material). With genomic information now revealing >1000 putative CLC homologs, we find that CLC-ck2 is not unique—several other uncharacterized homologs also lack these regions. To our knowledge, ours is the first study to characterize the function of a homolog missing these regions.Using Cl⁻ flux assays, we first sought to determine whether CLC-ck2 could catalyze Cl⁻ transport (10). With CLC-ck2-containing vesicles, slow but significant Cl⁻ efflux was observed upon addition of valinomycin (Vln; Fig. 1 A, blue trace). In control vesicles lacking CLC-ck2, no significant Cl⁻ flux was observed (Fig. 1 A, black). The CLC-ec1 inhibitor 4,4′-octanamidostilbene-2,2′-disulfonate (OADS) (11) completely inhibited Cl⁻ flux (Fig. 1 A, green). The Cl⁻ unitary turnover rate for wild-type CLC-ck2 was 31 ± 5 s⁻¹ (mean ± SE, n = 5). This rate is ∼2 orders of magnitude less than the Cl⁻ flux through the CLC-ec1 antiporter, and is much slower transport than expected for an ion channel. However, it is a similar to the rate catalyzed by the cyanobacterium antiporter CLC-sy1 (4).Open in a separate windowFigure 1(A) Representative Cl⁻ flux assays. Cl⁻ efflux was initiated by addition of Vln. Triton X-100 was added to disrupt liposomes and release all intracellular Cl⁻. The insert shows an expanded view of the efflux immediately after addition of Vln. (B) Representative H⁺ flux assays demonstrate Cl⁻-driven H⁺ influx. H⁺ flux was initiated by the addition of Vln. The H⁺ gradient was collapsed at the end by the addition of FCCP.To test whether CLC-ck2 is a Cl⁻ ion channel or Cl⁻/H⁺ antiporter, we performed H⁺ flux assays as previously described (10). If vesicles contain a Cl⁻/H⁺ antiporter, the efflux of Cl⁻ upon addition of Vln will drive the movement of protons into the vesicles against their concentration gradient. If vesicles contain a Cl⁻ ion channel, however, no movement of protons will be observed upon addition of Vln. We found that CLC-ck2 showed significant Cl⁻-driven H⁺ uptake. Fig. 1 B illustrates uphill movement of protons in the presence of a Cl⁻ gradient. H⁺ influx, like Cl⁻ efflux, was inhibited by the presence of OADS. These assays are not quantitative enough to determine Cl⁻/H⁺ stoichiometry. However, they qualitatively demonstrate that CLC-ck2 acts as a Cl⁻/H⁺ antiporter even though it lacks Glu_in.If Glu_in is important for maximizing H⁺ flux, we would expect that introducing a glutamate at the Glu_in position would increase the H⁺ flux observed through CLC-ck2. However, we found that the I175E mutation did not significantly alter H⁺ or Cl⁻ flux (Fig. 2). Hence, Glu_in does not enhance H⁺ transport through CLC-ck2.Open in a separate windowFigure 2Unitary turnover rates, calculated from initial velocities after addition of Vln in (A) Cl⁻ and (B) H⁺ flux assays. Reconstitutions contained 5–38 μg protein/mg lipid. Bars represent the mean ± SE for three to 17 assays.The external glutamate gate Glu_ex is conserved and required for H⁺ transport in all known CLC antiporters (5,7). To determine whether Glu_ex is also essential for H⁺ transport in CLC-ck2, we made the E122Q mutation. This mutant can still transport chloride but fails to move protons (Fig. 2). This mutant protein was not very stable in micelles, precipitating over the course of hours, and thus the unitary turnover rates shown in Fig. 2 represent lower limits. Nevertheless, this result is consistent with observations in other CLC antiporters and suggests that Glu_ex is important for H⁺ transport in all CLC antiporters.Because CLC-ck2 lacks amino acids that coordinate the Cl⁻ ions in the structure of CLC-ec1, we wondered whether the ion selectivity might differ. Indeed, the plant atCLC-a homolog has a single change in this region that makes it selective for NO₃⁻ over Cl⁻ (12). To determine the ion selectivity of CLC-ck2, we used radioactive uptake assays (11). In these assays, the amount of ³⁶Cl⁻ exchanged into CLC-ck2-containing vesicles loaded with cold Cl⁻ is measured as a function of time. Various anions were added to the extravesicular solution to test which ions were transported in preference to the ³⁶Cl⁻. A decrease in radioactive uptake indicates that the anion is permeant and/or blocks CLC-ck2. Fig. 3 A plots the amount of ³⁶Cl⁻ uptake with each of the various ions added; the ion selectivity (or block) was SO₄²⁻ ≫ Cl⁻ > NO₃⁻ > SCN⁻ >Br⁻ > F⁻ > P_i⁻ ≈ I⁻ ≫ isethionate⁻. This selectivity is similar to that of CLC-ec1 (13), with one noticeable exception: SO₄²⁻. SO₄²⁻ had no effect on CLC-ec1, but strongly competed with Cl⁻ uptake through CLC-ck2 (Fig. 3 B). Hence, the selectivity filter of CLC-ck2 is similar enough to other CLCs to transport Cl⁻, NO₃⁻, and Br⁻ as expected. However, further investigation is required to determine the structural differences that must underlie the distinct disparity in SO₄²⁻ permeability and/or block.Open in a separate windowFigure 3Ion selectivity of CLC-ck2. (A) Liposomes reconstituted with CLC-ck2 were screened for selectivity against various test ions in the presence of 1 mM ³⁶Cl⁻ at pH 4.5. All test ions were present at 10 mM, except for isethionate, which was present at 20 mM to confirm that it is inert. After 10 min, the radioactivity counts were measured to determine total ³⁶Cl⁻ uptake (for isethionate, uptake was stopped after 20 min). Counts were normalized with respect to liposome uptake in the absence of an external test ion. Bars represent the mean ± SE for three assays. (B) Comparison of effects of external sulfate on CLC-ec1 and CLC-ck2 on radioactive update assays, normalized as in part A.This study reveals that Glu_in is not essential for Cl⁻- coupled H⁺ transport in CLC-ck2, in direct contrast to the previous conclusion that the protonatable side chain of the glutamate is directly involved in the H⁺ transport pathway (14). Thus, our result brings into question the location of the H⁺ permeation pathway. The protons must be transferred via other protonatable residues or water molecules. The residue adjacent to Glu_in (E202 in CLC-ec1) is conserved in CLC-ck2. Unfortunately, mutation of this glutamate (E174F) in CLC-ck2 resulted in unstable protein that could not be characterized in functional studies. Using the structure of CLC-ec1 as a guide, we see no other obvious protonatable residues in CLC-ck2 available to transfer protons from the intracellular side to Glu_ex. One possibility is that H⁺ transport may require a water wire. The idea of a water wire is not new. In CLC-ec1, there is an ∼15 Å gap between Glu_in and Glu_ex, and it has never been clear exactly how protons cross this gap. Recent molecular-dynamics studies have supported the idea that the Glu_in in CLC-ec1 may help to position water molecules for a water wire to transfer protons to the extracellular glutamate (15). If indeed the role of Glu_in is simply to position water molecules properly to transfer protons, subtle changes in other parts of the structure could allow this water wire to exist in the absence of Glu_in. This could also explain how the eukaryotic CmCLC homolog, which has a threonine at the Glu_in position, is able to act as a coupled transporter as well. We have not yet been able to determine the structure of CLC-ck2 to understand how the lack of conserved amino acids near the Cl⁻-binding sites affects the structure. This study will inspire future work to investigate the molecular mechanism of CLC-ck2 and CLC-ck2 homologs in greater detail.     

High-performance liquid chromatographic analysis of mibefradil in dog plasma and urine

The objective of the study was to develop a sensitive and specific assay for studying the pharmacokinetics of a novel calcium antagonist, a benzimidazolyl-substituted tetraline derivative, mibefradil (I) in the dog. The assay involves liquid-liquid extraction of a biological sample, reversed-phase HPLC separation and fluorescence detection (λ_ex = 270 nm and λ_em = 300 nm) of a sample components. Each sample was eluted with a mobile phase pumping at a flow-rate of 2 ml/min. The mobile phase composition was a mixture of acetonitrile and aqueous solution (38:62, v/v). The aqueous solution contains 0.0393 M KH₂PO₄ and 0.0082 M Na-pentanesulphonic acid. The retention times were 10.7 min for I, and 12.2 min for internal standard Ro 40–6792. Calibration curves with concentrations of I ranging from 10 to 500 ng/ml were linear (r² > 0.99). The detection limit for I was 0.5 ng/ml when 0.5 ml of plasma or urine was used. Intra- and inter-day accuracy and precision were within 10%. The assay was successfully applied to the pharmacokinetic studies of I in dogs.     

The decomposition of triphenylimidazole‐para‐acetate follows specific base catalysis and can be conveniently followed by fluorescence

The kinetics of the decomposition reaction of 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenyl acetate ( 1 ) in basic alcoholic media was investigated, using a simple fluorescence (FL) spectrophotometric procedure. The process was conveniently studied using FL, since the triphenylimidazole‐derived ester 1 and its reaction products (the corresponding phenol 2 and phenolate 2 ^? ) are all highly fluorescent (Φ_FL > 37%). By carefully selecting excitation and emission wavelengths, observed rate constants k₁ in the order of 10^?3 to 10^?2 s^?1 were obtained from either reactant consumption (λ_ex = 300 nm, λ_em = 400 nm) or product formation (λ_ex = 350 nm, λ_em = 475 nm); these were shown to be kinetically equivalent. Intensity‐decay time profiles also gave a residual FL intensity parameter, shown to be associated to the distribution of produced species 2 and 2 ^? , according to the basicity of the medium. Studying the reaction in both methanol (MeOH) and isopropanol (iPrOH), upon addition of HO^?, provided evidence that the solvent's conjugate base is the active nucleophilic species. When different bases were used (tBuO^?, HO^?, DBU and TEA), bimolecular rate constants k_bim ranging from 4.5 to 6.5 L mol^?1 s^?1 were obtained, which proved to be non‐dependent on the base pK_aH, suggesting specific base catalysis for the decomposition of 1 in alcoholic media.     

Spectroscopic measurements of the parameters of the helium plasma jets generated in the plasma focus discharge at the PF-3 facility

The spectroscopic technique used to measure the parameters of the plasma jets generated in the plasma focus discharge and those of the plasma of the immobile gas through which these jets propagate is described. The time evolution of the intensities and shapes of spectral lines in experiments carried out with helium at the PF-3 facility was studied by means of electron-optical streak cameras. The plasma electron temperature, T ≈ 4–5 eV, was determined from the intensity ratio of two spectral lines, one of which (λ₁ = 5876 Å) belongs to neutral helium, while the other (λ₂ = 4686 Å), to hydrogen-like helium ions. The plasma density at different time instants was determined from the Stark broadening of these lines in the electric fields of different nature. The plasma density is found to vary from 4 × 10¹⁴ to 2 × 10¹⁷ cm^?3.     

Reversed-phase high-performance liquid chromatography of radioiodinated salmon calcitonins

Reversed-phase HPLC conditions for simultaneous separation of salmon calcitonin, mono- and di-radioiodinated salmon calcitonins and their tryptic digested fragments have been developed. Salmon calcitonin was radioiodinated with Na¹²⁵I by the iodo-beads method. After solid-phase extraction from the reaction mixtures using C₁₈ Bond Elut cartridges, mono- and di-radioiodinated salmon calcitonins were separated from each other, as well as from unlabeled salmon calcitonin, on a Bondclone 10 C₁₈ column (300×7.8 mm I.D.) by isocratic elution with 0.1% trifluoroacetic acid in 34% aqueous acetonitrile. The characteristics of either iodinated peptides or unlabeled salmon calcitonin were evaluated on the basis of UV absorbance (215 and 280 nm), fluorescence (λ_ex=282 nm, λ_em=310 nm) and measurement of specific radioactivity by means of a flow-through radio-isotope detector. HPLC separation of a tryptic digest of iodinated salmon calcitonin fraction on a W-porex 5 C₁₈ 300 Å column (250×4.6 mm I.D.) and subsequent amino acid analysis, led to the conclusion that radioiodination took place at the Tyr residue and not at the His moiety.     

Properties of the phenotypic variants of Pseudomonas aurantiaca and P. fluorescens

Different capacity for phenotypic variation of Pseudomonas aurantiaca and P. fluorescens in populations of cyst-like resting cells (CRC) during their germination on solid media, was shown to be a characteristic trait of biodiversity for the dormant forms of these bacteria. This biodiversity manifests itself as qualitative and quantitative differences in the spectra and emergence frequency of phenotype variants, obtained by plating of CRC, and depends on the conditions of CRC formation and storage time. In P. aurantiaca, the variation was associated with transition of the wild-type S-colonial phenotype into the R-type or the more pigmented P-type. These transitions were most pronounced for the CRC obtained under nitrogen depletion (a twofold N limitation), as well as under the influence of a chemical analogue of microbial anabiosis autoinducers, C₁₂-AHB. In the latter case, the frequency of S?R and S?P transitions (up to 70% and 80%, respectively) depended on the C₁₂-AHB concentration (1.0 × 10^?4 M and 2.5 × 10^?4 M) and on the storage time of CRC suspensions (from 3 days to 1.3 months). In the CRC populations grown in nitrogen-deficient media, R-type appeared with a frequency of up to 45% after at least four months of storage. In the case of P. fluorescens, S?R transitions depended not only on the storage time of CRC and C₁₂-AHB concentrations, but also on the composition of the solid medium used for plating. Differences were shown between the R-, P-, and S-variants of P. aurantiaca in such morphological, physiological, and biochemical characteristics as the growth rate (μ_max) in a poor medium, biomass yield (Y _max), resistance to streptomycin and tetracycline (LD₅₀), and the productivity in extracellular proteases. The R-and S-variants of P. fluorescens differed in their growth characteristics, resistance to high salinity and oxidative stress, as well as in their sensitivity to exogenous introduction of chemical analogues of microbial autoregulators (C₁₂-AHB and C₇-AHB). Hence, both the formation of dormant forms of the various morphological types [1] and intrapopulation phenotypic variability observed during their germination are important for the survival strategy of pseudomonads under unfavorable environmental conditions.     

Superoxide dismutase: a photochemical augmentation assay.

Cell envelope vesicles containing bacteriorhodopsin, prepared from Halobacterium halobium, have previously been shown to accumulate glutamate to high concentration gradients when illuminated. This active transport is energized by a sodium gradient (Na_out⁺ ? Na_in⁺), which arises from Na⁺-efflux coupled to the light-induced H⁺-gradient. The oxidation of dimethyl phenylenediamine (DPD) by the vesicles also can drive uphill glutamate transport, and such transport is inhibited by KCN, azide, ionophores, or uncouplers. K_T for glutamate is 1.4 × 10^?7m under these conditions, as compared to 1.3 × 10^?7m for light-induced transport. The respiration-induced transport of glutamate is dependent on high Na⁺ concentrations on the vesicle exterior and requires low Na⁺ concentrations in the interior. When Na⁺ of increasing concentrations is included in the vesicles, transport proceeds with increasing lags, similarly to the case of light-driven transport. In vesicles to which DPD is added first, and then KCN at increasing time intervals (5 to 15 min), glutamate transport occurs after the addition of KCN, with increasing rates, even though respiration is inhibited. This indicates that the energy generated by DPD-oxidation is conserved over several minutes. These results suggest that in the case of respiration-dependent glutamate transport the translocation is also driven by a Na⁺-gradient; thus, there is a single glutamate transport system independent of the source of energy. The generation of such an Na⁺-gradient during DPD-oxidation implies that the respiration component involved, cytochrome oxidase, is functionally equivalent to bacteriorhodopsin, which acts as a proton pump.     

A simple method for simultaneous RP-HPLC determination of indolic compounds related to bacterial biosynthesis of indole-3-acetic acid

In this short technical report, we present a fast and simple procedure for sample preparation and a single-run Reversed Phase High Performance Liquid Chromatography (RP-HPLC) determination of seven indoles (indole-3-acetic acid, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-lactic acid, tryptamine and tryptophan) in bacterial culture supernatants. The separation of the analytes, after a single centrifugal filtration clean-up step, was performed using a gradient elution on a symmetry C8 column followed by fluorimetric detection (λ_ex = 280/λ_em = 350 nm). The calibration curves were linear for all of the studied compounds over the concentration range of 0.0625–125 μg mL^?1 (r ²  ≥ 0.998) and the limits of detection were below 0.015 μg mL^?1. The applicability of the method was confirmed by analysis of Pseudomonas putida culture supernatants.     

Studies on chemiluminescence in the enzymatic and autoxidative transformation of 3,4-dihydroxyphenylalanine into eumelanins

The catechol oxidase-catalysed and autoxidative transformation of 3,4-dihydroxyphenylalanine (DOPA) to eumelanin have been studied by oxygen consumption, energy transfer, absorption and fluorescence spectroscopy. Formation of transient dopachrome (λ_max = 480 nm) and dopalutin (λ_ex = 423 nm, λ_em = 491 nm) have been found in the enzymatic and autoxidative reaction. In the enzymatic reaction, neither a photon emission with quantum yield Φ > 10^?13 nor energy transfer to triplet and singlet energy acceptors (sensitizers such as anthracene derivatives, xanthene dyes and chlorophyll-a) in water and micellar solutions have been found. The autoxidative reaction is chemiluminescent (Φ = 10^?9), the emission occurring in the 400-600 nm range. The excitation energy is not transferred to sensitizers. The effect of various enzymes and traps of active oxygen species as well as the spectral distribution of chemiluminescence indicate that there is no emission from oxygen dimoles. Carbonates and active species of oxygen are shown to participate in the chemiexcitation reaction.     

Insulin-regulated Glut4 Translocation: MEMBRANE PROTEIN TRAFFICKING WITH SIX DISTINCTIVE STEPS*

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (k_en = 0.2 min⁻¹ versus 0.6 min⁻¹). Differentiation decreases Glut4 k_en 40% (k_en = 0.12 min⁻¹). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (k_ex = 0.025–0.038 min⁻¹), not through the fast Tf receptor pathway (k_ex = 0.2 min⁻¹). The k_ex measured in adipocytes after insulin stimulation is similar (k_ex = 0.027 min⁻¹). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (k_sort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (k_seq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (k_fuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs.