Testicular expression of PC4 in the rat: molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase.

The rat cDNA sequence of PC4 (rPC4), representing a new member of the Kex2/subtilisin-like proprotein convertases, demonstrated the presence of at least three rPC4 mRNAs resulting in the production of rPC4-A (654 amino acids), rPC4-B (619 amino acids), and rPC4-C (609 amino acids) with different C-terminal sequences. Analogous to rat PC4, three cDNAs were also found for the mouse PC4. The observed molecular diversity of PC4 mRNA possibly results from the differential splicing and/or exon skipping of the parent gene. PC4 mRNA, with a major form at 2.8 kilobases, was highly abundant in the rat testis but could not be detected by Northern analysis in any other tissues including the central nervous system and peripheral tissues. Testicular cell separation studies combined with Northern analysis indicate the high expression levels of PC4 in germ cells but not in Leydig, Sertoli, or peritubular cells. In situ hybridization histochemistry confirms the site of PC4 gene expression as the pachytene spermatocytes and the round spermatids but not in the elongating spermatids. We also demonstrate the colocalization of PC4 with proenkephalin in testicular germ cells by in situ hybridization. A study of the ontogeny of PC4 indicated that PC4 mRNA was first expressed postnatally between days 19 and 22, coinciding with the first stages of spermiogenesis. The stage-specific expression of PC4 in testis indicates its potential role in the developmental maturation of germ cells and that this convertase may play a specific physiological function in reproduction.     

New strategies for the isolation and activity determination of naturally occurring type-4 glutathione peroxidase

Type 4 glutathione peroxidase (GPx4) is a widely expressed mammalian selenoenzyme known to play a vital role in cytoprotection against lipid hydroperoxide (LOOH)-mediated oxidative stress and regulation of oxidative signaling cascades. Since prokaryotes are not equipped to express mammalian selenoproteins, preparation of recombinant GPx4 via commonly used bacterial transformation is not feasible. A published procedure for isolating the enzyme from rat testis employs affinity chromatography on bromosulfophthalein–glutathione-linked agarose as the penultimate step in purification. Since this resin is no longer commercially available and preparing it in satisfactory operational form is tedious, we have developed an alternative purification approach based on sequential anion exchange, size exclusion, and cation exchange chromatography. Final preparations were found to be essentially homogeneous in GPx4 (M_r  20 kDa), as demonstrated by SDS–PAGE with protein staining and immunoblotting. Specific enzymatic activity was determined using a novel thin-layer chromatographic approach in which the kinetics of phosphatidylcholine hydroperoxide loss or cholesterol-7α-hydroperoxide loss was monitored. A >400-fold purification of active enzyme has been attained. The relatively straightforward isolation procedure described should prove valuable for further functional studies on GPx4, e.g. how its ability to catalyze LOOH reduction compares with that of other LOOH detoxifying enzymes.     

A [4+2] mixed ligand approach to ruthenium DNA metallointercalators [Ru(tpa)(N–N)](PF6)2 using a tris(2-pyridylmethyl)amine (tpa) capping ligand

A series of five tris(2-pyridylmethyl)amine (tpa) ruthenium complexes [Ru(tpa)(N–N)](PF₆)₂ with N–N = bpy (2,2′-bipyridine), phen (1,10-phenanthroline), dpq (dipyrido[3,2-d:2′,3′-f]quinoxaline), dppz (dipyrido[3,2-a;2′,3′-c]phenazine), and dppn (4,5,9,16-tetraazadibenzo[a,c]naphthacene) was prepared and characterized by NMR, UV–Visible (UV/Vis), and fluorescence spectroscopy as well as cyclic voltammetry. Structures optimized with density functional theory methods (DFT, BP86, TZVP) without constraints show C₁ symmetry while in solution, the ¹H and ¹³C NMR spectra are in accordance with an average C_s symmetry. This is thought to be due to a low energy barrier for flipping of the equatorial pyridine ring from one side of the N–N plane to the other. The electronic structure of the compounds was studied with DFT and a change in the highest occupied molecular orbital (HOMO) character from Ru t_2g for the bpy, phen, and dpq to N–N ligand-based for the dppz and dppn complexes was found. TDDFT calculations showed dominant N–N-based intra-ligand charge transfer (ILCT) transitions in the latter two complexes mixed with metal-to-ligand charge transfer (MLCT) bands found for all five compounds. DNA binding of the complexes was studied with UV/Vis titrations, the fluorescent ethidium bromide displacement assay, and CD spectroscopy. The affinity increases with the aromatic surface area of of the bidentate N–N ligand in the order bpy  phen < dpq < dppz  dppn. Viscosity measurements support an intercalative binding mode for the latter three compounds, while the others did not show a pronounced effect of the hydrodynamic properties of calf thymus (CT) DNA.     

Biphasic kinetics in the reaction between amino acids or glutathione and the chromium acetate cluster, [Cr3O(OAc)6]

Kinetics for the breakdown of the trinuclear chromium acetate cluster with a series of monoprotic and diprotic amino acid ligands and with glutathione in aqueous media have been investigated spectrophotometrically at pH 3.5–5.5 and in a temperature range of 45–60 °C. Under pseudo-first-order conditions, reactions with these ligands exhibited biphasic kinetic behavior that can be accounted for by a consecutive two-step reaction, A → B → C, where A is assumed to be a forced ion pair, B an intermediate and C is the product; experimental data fit to a biexponential equation for the transformation. Rates for k_short, k_long, and k_obs were determined by manual extrapolation of absorbance data or curve-fitting routines; associated activation parameters for each step of the reaction were calculated using the Eyring equation. Rates for the first and second steps of the reaction are on the order of 10⁻⁴ and 10⁻⁵ s⁻¹, respectively. The large negative values of ΔS^‡ and smaller ΔH^‡ in the first step indicate an associative step, while high positive values of ΔS^‡ in the second step indicate dissociation. To account for the results mechanistically, the results are interpreted to be a first step of ligand exchange with a pseudo-axial aqua ligand, followed by a dissociative step involving acetate or oxo ligand displacement. The dissociative step is the rate determining step, with k_obs ≈ k_long.The results demonstrate reaction pathways that are available to the Cr(III) metal centers that may be physiologically relevant in the ligand-rich environment of biological systems. Under general conditions Cr(III) clusters may be expected to be broken down, unless some unique biological environment stabilizes the cluster. The present study has application to the processes related to Cr(III) transport and excretion, to potential mechanisms of Cr(III) action in a biological setting, and to the pharmacokinetics of Cr(III) supplements for animal and human consumption.     

Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Enteropeptidase (enterokinase EC 3.4.21.9), catalyzing trypsinogen activation, exhibits unique properties for high efficiency hydrolysis of the polypeptide chain after the N-terminal tetraaspartyl-lysyl sequence. This makes it a convenient tool for the processing of fusion proteins containing this sequence. We found the enteropeptidase-catalysing degradation of some bioactive peptides: cattle hemoglobin beta-chain fragments Hb (2–8) (LTAEEKA) and Hb (1–9) (MLTAEEKAA), human angiotensin II (DRVYIHPF) (AT). Model peptideswith truncated linker WDDRG and WDDKG also were shown to be susceptible to enteropeptidase action. Kinetic parameters ofenteropeptidase hydrolysis for these substrates were determined.K _m values for all substrates with truncated linker (10^-3 M) are an order of magnitude higher thancorresponding values for typical enteropeptidase artificial peptide or fusion protein substrates with full enteropeptidase linker –DDDDK– (K _m 10^-4 M). k _cat values for AT, Hb (2–8), WDDRG and WDDKG are 30–40 min^-1. But one additional amino acid residue at both N- and C-terminus of Hb (2–8) results in a drastic increase of hydrolysis efficiency: k _cat value for Hb (1–9) is 1510 min^-1. Recent study demonstrates the possibility of undesirable cleavage of target peptides or proteins containing the above-mentioned truncated linker sequences; further, the ability of enteropeptidase to hydrolyse specifically several biologically active peptides in vitro along with its unique natural substrate trypsinogen was demonstrated.     

Comparative study of the binding pockets of mammalian proprotein convertases and its implications for the design of specific small molecule inhibitors

Proprotein convertases are enzymes that proteolytically cleave protein precursors in the secretory pathway to yield functional proteins. Seven mammalian subtilisin/Kex2p-like proprotein convertases have been identified: furin, PC1, PC2, PC4, PACE4, PC5 and PC7. The binding pockets of all seven proprotein convertases are evolutionarily conserved and highly similar. Among the seven proprotein convertases, the furin cleavage site motif has recently been characterized as a 20-residue motif that includes one core region P6-P2´ inside the furin binding pocket. This study extended this information by examining the 3D structural environment of the furin binding pocket surrounding the core region P6-P2´ of furin substrates. The physical properties of mutations in the binding pockets of the other six mammalian proprotein convertases were compared. The results suggest that: 1) mutations at two positions, Glu230 and Glu257, change the overall density of the negative charge of the binding pockets, and govern the substrate specificities of mammalian proprotein convertases; 2) two proprotein convertases (PC1 and PC2) may have reduced sensitivity for positively charged residues at substrate position P5 or P6, whereas the substrate specificities of three proprotein convertases (furin, PACE4, and PC5) are similar to each other. This finding led to a novel design of a short peptide pattern for small molecule inhibitors: [K/R]-X-V-X-K-R. Compared with the widely used small molecule dec-RVKR-cmk that inhibits all seven proprotein convertases, a finely-tuned derivative of the short peptide pattern [K/R]-X-V-X-K-R may have the potential to more effectively inhibit five of the proprotein convertases (furin, PC4, PACE4, PC5 and PC7) compared to the remaining two (PC1 and PC2). The results not only provide insights into the molecular evolution of enzyme function in the proprotein convertase family, but will also aid the study of the functional redundancy of proprotein convertases and the development of therapeutic applications.     

Optimization of cell density and dilution rate in <Emphasis Type="Italic">Pichia pastoris</Emphasis> continuous fermentations for production of recombinant proteins

This paper provides an approach for optimizing the cell density (X_c) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon (INF-). The objective was to maximize the volumetric productivity (Q, mg INF- l^–1 h^–1), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X_c and D within the ranges 150 X_c 450 g cells (wet weight) l^–1 and 0.1 _mD0.9 _m (_m=0.0678 h^–1, the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired X_c and D were determined based on the mass balance. From the RSM model, the optimal X_c and D were 328.9 g l^–1 and 0.0333 h^–1 for a maximum Q of 2.73 mg l^–1 h^–1. The model of specific production rate (, mg INF- g^–1 cells h^–1) was also established and showed the optimal X_c=287.7 g l^–1 and D=0.0361 h^–1 for the maximum (predicted to be 8.92×10^–3 mg^–1 g^–1 h^–1). The methanol specific consumption rate (, g methanol g^–1 cells h^–1) was calculated and shown to be independent of the cell density. The relationship between and (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.     

Effect of tree distance on N2O and CH4-fluxes from soils in temperate forest ecosystems

Complete annual cycles of N₂O and CH₄ flux in forest soils at a beech and at a spruce site at the Höglwald Forest were followed in 1997 by use of fully automatic measuring systems. In order to test if on a microsite scale differences in the magnitude of trace gas exchange between e.g. areas in direct vicinity of stems and areas in the interstem region at both sites exist, tree chambers and gradient chambers were installed in addition to the already existing interstem chambers at our sites. N₂O fluxes were in a range of –4.6–473.3 g N₂O-N m^–2 h^–1 at the beech site and in a range of –3.7–167.2 g N₂O-N m^–2 h^–1 at the spruce site, respectively. Highest N₂O emissions were observed during and at the end of a prolonged frost period, thereby further supporting previous findings that frost periods are of crucial importance for controlling annual N₂O losses from temperate forests. Fluxes of CH₄ were in a range of +10.4––194.0 g CH₄ m^–2 h^–1 at the beech site and in a range of –4.4––83.5 g CH₄ m^–2 h^–1 at the spruce site. In general, both N₂O-fluxes as well as CH₄-fluxes were higher at the beech site. On a microsite scale, N₂O and CH₄ fluxes at the beech site were highest within the stem area (annual mean: 49.6±3.3 g N₂O-N m^–2 h^–1; –77.2±3.1 g CH₄ m^–2 h^–1), and significantly lower within interstem areas (18.5±1.4 g N₂O-N m^–2 h^–1; –60.2±1.8 g CH₄ m^–2 h^–1). Significantly higher values of total N, C and pH in the organic layer, as well as increased soil moisture, especially in spring, in the stem areas, are likely to contribute to the higher N₂O fluxes within the stem area of the beech. Also for the spruce site, such differences in trace gas fluxes could be demonstrated to exist (mean annual N₂O emission within (a) stem areas: 9.7±0.9 g N₂O-N m^–2 h^–1 and (b) interstem areas: 6.2±0.6 g N₂O-N m^–2 h^–1; mean annual CH₄ uptake within (a) stem areas: –26.1±0.6 g CH₄ m^–2 h^–1 and (b) interstem areas: –38.4±0.8 g CH₄ m^–2 h^–1), though they were not as pronounced as at the beech site.     

Modulation of allo-immune responsesin vivo andin vitro by sperm specific lactate dehydrogenase-C4

The role of sperm specific lactate dehydrogenase-C₄ (LDH-C₄) in allo-immune responses using mixed lymphocyte cultures (MLC) and cytotoxic T cell (CTL) generationin vitro and local graft versus host (LGVH) reaction and allograft enhancementin vivo has been ascertained. LDH was purified from testes (LDH-C₄) and kidney (LDH-B₄) of C57 Bl/Ks mice. MLC and CTL were performed using C57 Bl/Ks-anti A/J lymphocytes in presence of 10^–3-1 g LDH-B₄ or LDH-C₄ per culture. The MLC and CTL responses showed biphasic action depending on the dose of LDH-C₄. Early MLC culture gave significantly low stimulation index at 10^–2–10^–1 g LDH-C₄ as compared to non-treated control cultures. However, the MLC response in presence of LDH-C₄ was not different from the LDH-B₄ treated one which showed a similar biphasic trend. On the other hand,⁵¹Cr release from YAC-222 target cells was practically abolished by LDH-C₄ at 10^–3–1^–1 g, and this was strikingly different from LDH-B₄ or non-treated cultures. LGVH reactivity as performed by using C57 Bl/Ks lymphocytes along with LDH-C₄ in (C57 Bl/Ks x A/J) F₁ hybrids indicated a suppression of stimulation index in primary and secondary (i.e. preimmunized in presence of LDH-C₄ or LDH-B₄) LGVH. Allograft enhancement of Sa I (A/J) in C57 Bl/Ks mice in presence of LDH-C₄, was delayed slightly but significantly during primary or secondary transplantation reaction. The reaction of LDH-C₄ in the modulation of allo-immune responses was more specificin vivo thanin vitro since the B₄ isozyme did not modify LGVH and Sa1 allograft rejection. Resultsper se suggest that LDH-C₄ is immunosuppressive for cell mediated allo-immune responsesin vivo andin vitro.     

Ammonium and nitrate uptake in gap,generalist and understory species of the genus Piper

Summary We studied root net uptake of ammonium (NH ₄ ⁺ ) and nitrate (NO ₃ ^– ) in species of the genus Piper (Piperaceae) under high, intermediate and low photosynthetically active photon flux densities (PFD). Plants were grown hydroponically, and then transferred to temperature controlled (25° C) root cuvettes for nutrient uptake determinations. Uptake solutions provided NH ₄ ⁺ and NO ₃ ^– simultaneously (both) or separately (single). In the first experiment, seven species of Piper, from a broad range of rainforest light habitats ranging from gap to understory, were screened for mineral nitrogen preference (100 M NH ₄ ⁺ and/or 100 M NO ₃ ^– ) at intermediate PFD (100 mol m^–2 s^–1). Preference for NH ₄ ⁺ relative to NO ₃ ^– , defined as the ratio of NH ₄ ⁺ (both):NO ₃ ^– (both) net uptake, was higher in understory species than in gap species. Ammonium repression of NO ₃ ^– uptake, defined as the ratio of NO ₃ ^– (single): NO ₃ ^– (both) net uptake, was also higher in understory species as compared to gap species. In a second set of experiments, we examined the effect of nitrogen concentration (equimolar, 10 to 1000 M) on NH ₄ ⁺ preference and NH ₄ ⁺ repression of NO ₃ ^– net uptake at high (500 mol m^–2 s^–1) and low (50 mol m^–2 s^–1) PFD in a gap (P. auritum), generalist (P. hispidum) and understory species (P. aequale). All species exhibited negligible NH ₄ ⁺ repression of NO ₃ ^– net uptake at high PFD. At low PFD, NH ₄ ⁺ preference and repression of NO ₃ ^– net uptake occurred in all species (understory > generalist > gap), but only at intermediate nitrogen concentrations, i.e. between 10 and 200 M. Ammonium repression of net NO ₃ ^– uptake decreased or increased rapidly (in < 48 h) after transitions from low to high or from high to low PFD respectively. No significant diurnal patterns in NO ₃ ^– or NH ₄ ⁺ net uptake were observed.CIWDPB publication # 1130     

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Use of 4-Thiouridine and 4-Thiothymidine in Studies on Pyrimidine Nucleoside Phosphorylases

The new substrates 4-thiouridine and 4-thiothymidine were proposed for spectrophotometric measurement of the activity of uridine (UP) and thymidine (TP) phosphorylases. At pH 7.5, 4-thiouridine has an absorbance maximum at 330 nm, and the difference in extinction coefficient () between 4-thiouridine and 4-thiouracil is 3000 ^–1cm^–1. 4-Thiouridine proved to be a good substrate for UP: the Michaelis ( ) and catalytic (k _cat) constants were estimated respectively at 130 M and 49 s^–1 at 25°C. Even a greater (5000 M^–1cm^–1 at 336 nm) was observed for the 4-thiothymidine/4-thiothymine pair.     

Development of reactor model for glucose isomerization catalyzed by whole-cell immobilized glucose isomerase

Based on the kinetic constants determined and the mathematical model of the reactor system developed, the performance of axial flow packed bed continuous enzyme reactor system was studied experimentally and also simulated with the aid of a computer for ultimate objective of optimization of the glucose isomerase reactor system.A reactor model was established analogous to heterogeneous catalytic reactor model taking into account the effect of fluid mass transfer and reversible kinetics. The investigated catalyst system consists of immobilized Streptomyces bambergiensis cells containing the enzyme glucose isomerase, which catalyzes the isomerization of glucose to fructose.List of Symbols A ₀, A ₁, A ₂ parameters in axial dispersion reactor model - c _go, c_g, c_gemol m^–3 glucose concentration at time t=0, at any time and at equilibrium conditions - c _gsmol m^–3 glucose concentration at particle surface - C dimensionless glucose concentration - d _pm particle diameter - d _rm diameter of reactor tube - Da Damkohler number - D _eff m² s^–1 effective glucose diffusion coefficient in Ca-alginate gel beads - k _fm s^–1 film transfer coefficient - K _e equilibrium constant - K _mg, K_mfmol m^–3 Michaelis-Menten constant for glucose and fructose, respectively - K _mmol m^–3 modified Michaelis-Menten constant - K dimensionless parameter - K ^* dimensionless parameter - L m length of reactor tube - Pe Peclet number - Pe _p particle Peclet number - Q m³ s^–1 volumetric flow rate - (-r _g) mol m^–3 s^–1 reaction rate - Re _p Reynolds particle number - Sc Schmidt number - Sh Sherwood number - t s time - v ₀ m s^–1 linear superficial fluid velocity - V _mg, V_mfmol g^–1 s^–1 maximal reaction rate for glucose and fructose, respectively - V _mmol m^–3 s^–1 modified maximal reaction rate for glucose - V _mg ^x mol m^–2 s^–1 maximal reaction rate for glucose - X _g, X_ge glucose conversion and glucose conversion at equilibrium conditions - X normalized conversion - Y dimensionless glucose concentration - void fraction of fixed bed - effectiveness factor of biocatalyst - Pa s kinematic viscosity of substrate - ₁ s first absolute weighted moment - ₂ s² second central weighted moment - _gkg m^–3 substrate density - _pkg m^–3 particle density - ² dimensionless variance of RTD curve - s residence time     

Trends in aluminium export from a mountainous area to surface waters, from deglaciation to the recent: Effects of vegetation and soil development, atmospheric acidification, and nitrogen-saturation

We reconstructed the history of terrestrial export of aluminium (Al) to Plešné Lake (Czech Republic) since the lake origin 12,600 year BC, and predicted Al export for 2010–2050 on the basis of previously published and new data on mass budget studies, palaeolimnological data, and MAGIC modelling. We focused on three major Al forms; ionic Al (Al_i), organically-bound Al (Al_o), and particulate Al hydroxide [Al(OH)₃]. In early post-glacial time, Plešné Lake received high terrestrial export of Al, but with a minor proportion of Al(OH)₃ (4–25 μM), and concentrations of Al_i and Al_o were negligible. Since the forest and soil development (9900–9000 year BC), erosion has declined and soil organic acids increased export of Al_o from soils. The terrestrial Al_o leaching (7.5 μM) persisted throughout the Holocene until the industrial period. Then, Al_i concentrations continuously increased (up to 28 μM in the mid-1980s) due to atmospheric acidification; the Al_i leaching was mostly associated with sulphate. The proportion of Al_i associated with nitrate has been increasing since the beginning of lake recovery from acidification after 1990 due to reduction in sulphur deposition and nitrogen-saturation of the catchment, leading to persistent nitrate leaching. Currently, nitrate has become the dominant strong acid anion and the major Al_i carrier. Al_o (5.5 μM) is predicted to dominate Al concentrations around 2050, but the predicted Al_i concentrations (4 μM) are uncertain because of uncertainty associated with the future nitrate leaching and its effect on soils.     

Action potential duration and force-frequency relationship in isolated rabbit,guinea pig and rat cardiac muscle

The effect of action potential duration and elevated cytosolic sodium concentration on the forcefrequency relationship in isolated rabbit, guinea pig and rat papillary muscle preparations was studied. Shortening of action potential duration in guinea pig and rabbit from 150–200 ms to values characteristic of rat (20–40 ms), using the K_(ATP) channel activator levkromakalim (15 mol·l^–1), markedly reduced the force of contraction and converted the positive force-frequency relationship into negative one at longer pacing cycle lengths. This conversion was greatly enhanced in the presence of acetylstrophanthidin (0.2–1 mol·l^–1), an inhibitor of the Na-K pump. Acetylstrophanthidin (1 mol·l^–1) alone, however, had no effect on the forcefrequency relationship. Prolongation of action potential duration in rat with inhibitors of cardiac K channels (4-aminopyridine [10 mmol·l^–1] plus tetraethylammonium [2 mmol·l^–1) increased the force of contraction and abolished the negative force-frequency relationship observed in rat at longer pacing-cycle lengths. It is concluded that both action potential duration and cytosolic sodium concentration are major determinants of the force-frequency relationship in mammalian myocardium.Abbreviations AC acetylstrophanthidin - APD action potential duration - APD ₅₀ and APD ₉₀ action potential duration measured at 50% and 90% level of repolarization, respectively - SR sarcoplasmic reticulum     

Seasonal variability of apoplastic NH4 + and pH in an intensively managed grassland

The stomatal compensation point of ammonia (_s) is a major factor controlling the exchange of atmospheric ammonia (NH₃) with vegetation. It is known to depend on the supply of nitrogen and to vary among plant species, but its seasonal variation has not yet been reported for grassland. In this study, we present the temporal variation of apoplastic NH₄ ⁺ concentration ([NH₄ ⁺]_apo) and pH (pH_apo) measured in leaves of Lolium perenne L. in a grassland, through two periods of cutting / fertilisation, followed by a livestock grazing period. The total free NH₄ ⁺ concentration measured in foliage ([NH₄ ⁺]_fol), and soil mineral NH₄ ⁺ and NO₃ ^– concentration are also presented. The value of [NH₄ ⁺]_apo varied from less than 0.01 mM to a maximum of 0.5 mM occurring just after fertilisation, whereas the apoplastic pH ranged from pH 6 to 6.5 for most of the time and increased up to pH 7.8, 9 days after the second fertilisation, when grazing started. [NH₄ ⁺]_fol varied between 20 and 50 g N-NH₄ ⁺ g^–1 f.w. The compensation point at 20°C, ranged from 0.02 g NH₃ m^–3 between the fertilisations to 10 g NH₃ m^–3 just after the second fertilisation. The reasons for these seasonal changes are discussed, with respect to plant metabolism and the concentration of ammonium and nitrate in the soil.     

Separation of proteins by ion-exchange chromatography using gradient elution

Chromatographic separation of proteins by the gradient elution method using DEAE Toyopearl 650^® was carried through. The concentration gradient was effected by changing the ionic strength of NaCl in the carrier buffer solution. Bovine serum albumin and hemoglobin were used as model proteins for separation. The experimental chromatogram was compared with theoretical results of Yamamoto et al. [1, 2]. Adsorption equilibria of the proteins onto the carrier were measured and expressed by a function of the ionic strength. The retention volume and peak width of the resulting chromatogram can be calculated from the equilibrium data using the Yamamoto theory. The calculated results agreed well with the experimental data.The method presented in this paper will be useful to predict the viability of ion-exchange chromatography in protein separation.List of Symbols c kg m^–3 concentration in the liquid phase - c _s kg m^–3 concentration in the solid phase - D _s m² s^–1 intraparticle diffusivity - d _p m particle diameter - E _z m² s^–1 longitudinal diffusivity of the protein - E _{z I} m² s^–1 longitudinal diffusivity of ionic strength - H /(1 – ) - I kmol m^–3 ionic strength - I _O kmol m^–3 initial ionic strength - I _p kmol m^–3 ionic strength at the peak - I _s kmol m^–3 ionic strength in the solid phase - I/V mol (dm³)^–2 slope of the ionic gradient elution - m distribution coefficient - m distribution coefficient at I - m _I distribution coefficient for ionic strength - Q cm³s^–1 flow rate - R m particle radius - R _s degree of separation - r m radial position inside particles - t s time - u m s^–1 linear velocity - V cm³ eluted volume of liquid - V _p cm³ eluted volume of liquid at the peak - V _T cm³ volume of the packed bed - W cm³ peak width - Z m bed height - z m vertical position in the bed - z _p m peak position from the inlet of the bed - (t) delta input at time - void fraction - ₁ s first moment - ₂ s² second central moment - s superficial space time     

Solution structure of T4moC,the Rieske ferredoxin component of the toluene 4-monooxygenase complex

Toluene 4-monooxygenase, a four-protein complex from Pseudomonas mendocina KR1, catalyzes the NADH- and O₂-dependent hydroxylation of toluene to form p-cresol. The solution structure of the 112-amino-acid Rieske ferredoxin component, T4moC, was determined from 2D and 3D ¹H, ¹³C, and ¹⁵N NMR data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space with input from 1650 experimental restraints, including 1264 inter-proton distance restraints obtained from NOEs, 247 non-redundant intra-residue NOEs, 26 hydrogen bond restraints, and 113 dihedral angle (, ) restraints. The 20 calculated conformers that best satisfied the input restraints were submitted to refinement in explicit solvent to improve the stereochemical quality. With exclusion of ill-defined N- and C-terminal segments (Ser2; His111–Ser112) and residues near to the [2Fe-2S] cluster, the atomic root mean square deviation for the 20 conformers with respect to the mean coordinates was 1.09 Å for the backbone and 1.60 Å for all non-hydrogen atoms. The T4moC structure consists of 10 -strands arranged in the three anti-parallel -sheet topology observed in all Rieske [2Fe-2S] domain proteins. The S of Cys45 and Cys64 and the N¹ of His47 and His67 provide the ligands to the [2Fe-2S] cluster of T4moC. ¹H–¹⁵N HSQC measurements show that both His47-N² and His67-N² are protonated at the pH of the NMR experiments. Comparisons are made between the present NMR structure, previous paramagnetic NMR studies of T4moC, and the X-ray structures of other members of the Rieske protein family.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0594-4     

Control of carbon mineralization to CH4 and CO2 in anaerobic,Sphagnum-derived peat from Big Run Bog,West Virginia

The mineralization of organic carbon to CH₄ and CO₂ inSphagnum-derived peat from Big Run Bog, West Virginia, was measured at 4 times in the year (February, May, September, and November) using anaerobic, peat-slurry incubations. Rates of both CH₄ production and CO₂ production changed seasonally in surface peat (0–25 cm depth), but were the same on each collection date in deep peat (30–45 cm depth). Methane production in surface peat ranged from 0.2 to 18.8 mol mol(C)^–1 hr^–1 (or 0.07 to 10.4 g(CH₄) g^–1 hr^–1) between the February and September collections, respectively, and was approximately 1 mol mol(C)^–1 hr^–1 in deep peat. Carbon dioxide production in surface peat ranged from 3.2 to 20 mol mol(C)^–1 hr^–1 (or 4.8 to 30.3 g(CO₂) g^–1 hr^–1) between the February and September collections, respectively, and was about 4 mol mol(C)^–1 hr^–1 in deep peat. In surface peat, temperature the master variable controlling the seasonal pattern in CO₂ production, but the rate of CH₄ production still had the lowest values in the February collection even when the peat was incubated at 19°C. The addition of glucose, acetate, and H₂ to the peat-slurry did not stimulate CH₄ production in surface peat, indicating that CH₄ production in the winter was limited by factors other than glucose degradation products. The low rate of carbon mineralization in deep peat was due, in part, to poor chemical quality of the peat, because adding glucose and hydrogen directly stimulated CH₄ production, and CO₂ production to a lesser extent. Acetate was utilized in the peat by methanogens, but became a toxin at low pH values. The addition of SO₄ ^2– to the peat-slurry inhibited CH4 production in surface peat, as expected, but surprisingly increased carbon mineralization through CH4 production in deep peat. Carbon mineralization under anaerobic conditions is of sufficient magnitude to have a major influence on peat accumulation and helps to explain the thin (< 2 m deep), old (> 13,000 yr) peat deposit found in Big Run Bog.     

Investigation of subcellular acidic compartments using alpha-aminophosphonate 31P nuclear magnetic resonance probes

The ³¹P nuclear magnetic resonance (NMR) characteristics, toxicity, and cellular penetration of five linear or cyclic α-aminophosphonate highly sensitive pH probes were investigated in Dictyostelium discoideum cells and isolated rat hearts and were compared with three phosphonic acid derivatives. The line width broadening at pH pK_a, which was satisfactorily modelized for all compounds, was significantly limited in biological milieu for the new markers, affording a four- to sixfold better accuracy in pH determination. Cellular uptake or washout of nontoxic concentrations (<15 mM) of α-aminophosphonates occurred by rapid passive permeation, whereas standard probes required a much slower fluid-phase pinocytosis and transport processes that could ultimately lead to trapping. Using mild concentrations (<4 mM) three α-aminophosphonates having 6 < pK_a < 7 allowed an easy and simultaneous ³¹P NMR determination of cytosolic, acidic, and extracellular compartments in anoxic–reoxygenated or starving D. discoideum.