Rates of intercalator-driven platination of DNA determined by a restriction enzyme cleavage inhibition assay

A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum–acridine agents represented by the formula [Pt(am₂)LCl](NO₃)₂, where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5′-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am₂ is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k _obs = 1.4 ± 0.37 × 10⁻⁴ s⁻¹ (t _1/2 = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k _obs = 5.7 ± 0.58 × 10⁻⁴ s⁻¹, t _1/2 = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k _obs = 2.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k _obs = 1.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine–purine cross-links are discussed.     

Stereoselective and driving-force-dependent photoinduced electron-transfer reactions of zinc myoglobin with optically active N,N′-dimethylcinchoninium and N,N′-dimethylcinchonidinium ions

In order to understand the detailed mechanism of the stereoselective photoinduced electron-transfer (ET) reactions of zinc-substituted myoglobin (ZnMb) with optically active molecules by flash photolysis, we designed and prepared new optically active agents, such as N,N′-dimethylcinchoninium diiodide ([MCN]I₂) and N,N′-dimethylcinchonidinium diiodide ([MCD]I₂). The photoexcited triplet state of ZnMb, ³(ZnMb)*, was successfully quenched by [MCN]²⁺ and [MCD]²⁺ ions to form the radical pair of ZnMb cation (ZnMb^·+) and reduced [MCN]^·+ and [MCD]^·+, followed by a thermal back ET reaction to the ground state. The rate constants (k _q) for the ET quenching at 25 °C were obtained as k _q(MCN)=(1.9±0.1)×10⁶ M⁻¹ s⁻¹ and k _q(MCD)=(3.0±0.2)×10⁶ M⁻¹ s⁻¹, respectively. The ratio of k _q(MCD)/k _q(MCN)=1.6 indicates that the [MCD]²⁺ preferentially quenches ³(ZnMb)*. The second-order rate constants (k _b) for the thermal back ET reaction from [MCN]^·+ and [MCD]^·+ to ZnMb^·+ at 25 °C were k _b(MCN)=(0.79±0.04)×10⁸ M⁻¹ s⁻¹ and k _b(MCD)=(1.0±0.1)×10⁸ M⁻¹ s⁻¹, respectively, and the selectivity was k _q(MCD)/k _q(MCN)=1.3. Both quenching and thermal back ET reactions are controlled by the ET step. In the quenching reaction, the energy differences of ΔΔH ^≠(MCD–MCN) and ΔΔS ^≠(MCD–MCN) at 25 °C were obtained as −1.1 and 0 kJ mol⁻¹, respectively. On the other hand, ΔΔH ^≠(MCD–MCN)=11±2 kJ mol⁻¹ and TΔΔS ^≠(MCD–MCN)=−10±2 kJ mol⁻¹ were given in the thermal back ET reaction. The highest stereoselectivity of 1.7 for [MCD]^·+ found at low temperature (10 °C) was due to the ΔΔS ^≠ value obtained in the thermal back ET reaction. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Stimulation and oxidative catalytic inactivation of thermolysin by copper•Cys-Gly-His-Lys

[Cu²⁺•Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 μM) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 μM, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 μM. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu²⁺–amino-terminal copper and nickel binding motif (λ _max ∼ 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu²⁺ charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (ΔE = 92 mV) for the Cu^3+/2+ redox couple obtained for [Cu²⁺•Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu²⁺•Lys-Gly-His-Lys]⁺ (0.84 V, ΔE = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu²⁺•Lys-Gly-His-Lys]⁺ homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O₂) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as k _obs = 7.7 × 10⁻² min⁻¹, yielding a second-order rate constant of (7.7 ± 0.9) × 10⁴ M⁻¹ min⁻¹. Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone.     

A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen

A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn²⁺, suppressed by Cu²⁺ and Zn²⁺ but inhibited by Fe³⁺. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 ± 6.1 U mg⁻¹ protein) by 4–10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 ± 74.7 U mg⁻¹ was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC₅₀ = 2.41 ± 0.03 versus 1.25 ± 0.01 mM) and conidial germination (EC₅₀ = 0.89 ± 0.06 versus 0.55 ± 0.07 mM), and 23% more tolerant to UV-B irradiation (LD₅₀ = 0.49 ± 0.02 versus 0.39 ± 0.01 J cm⁻²). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT₅₀ = 4.5 (4.2–4.8) versus 5.7 (5.2–6.4) days]. Our study highlights for the first time that the Mn²⁺-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.     

Purification and Characterization of an Esterase from <Emphasis Type="Italic">Acinetobacter lwoffii</Emphasis> I6C-1

EstA was purified from the supernatant by A. lwoffii 16C-1. Its molecular mass was determined to be 45 kDa, and the optimal activity occurred when the pH level was 8.0 at a temperature of 37°C. The activation energies for the hydrolysis of p-nitrophenyl butyrate was determined to be 11.25 kcal/mol in the temperature range of 10–37°C. The enzyme was unstable at temperatures higher than 50°C. The Michaelis constant (K _m ) and V _max for p-nitrophenyl butyrate were 11 μM and 131.6 μM min⁻¹ mg of protein-1, respectively. The enzyme was strongly inhibited by Hg²⁻, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, ethylemediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP). Received: 20 August 2001 / Accepted: 20 September 2001     

P2Y(2) receptor-mediated inhibition of amiloride-sensitive short circuit current in M-1 mouse cortical collecting duct cells

Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y₂ receptors stimulate luminal Ca²⁺-activated Cl⁻ channels and inhibit Na⁺ transport. Here we address the mechanism of ATP-mediated inhibition of Na⁺ transport. M-1 cells had a transepithelial voltage (V _te ) of −31.4 ± 1.3 mV and a transepithelial resistance (R _te ) of 1151 ± 28 Ωcm². The amiloride-sensitive short circuit current (I _sc ) was −28.0 ± 1.1 μA/cm². The ATP-mediated activation of Cl⁻ channels was inhibited when cytosolic Ca²⁺ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca²⁺]_i increase was paralleled by a rapid and transient R _te decrease (297 ± 51 Ωcm²). In the presence of CPA, basolateral ATP led to an R _te increase by 144 ± 17 Ωcm² and decreased V _te from −31 ± 2.6 to −26.6 ± 2.5 mV. I _sc dropped from −28.6 ± 2.4 to −21.6 ± 1.9 μA/cm². Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na⁺ absorption. Lowering [Ca²⁺]_i by removal of extracellular Ca²⁺ did not alter the ATP effect. PKC inhibition or activation were without effect. Na⁺ absorption was activated by pH_i alkalinization and inhibited by pH_i acidification. ATP slightly acidified M-1 cells by 0.05 ± 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y₂ receptors inhibits Na⁺ absorption. This effect is not mediated via [Ca²⁺]_i, does not involve PKC and is to a small part mediated via intracellular acidification. Received: 9 February 2001/Revised: 17 May 2001     

Chromate reduction by Rhodobacter sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K _m of 15±1.3 μM CrO₄ ²⁻ and a V _max of 420±50 μmol min⁻¹ mg protein⁻¹ was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO₄ ²⁻ and 100±9.6 μmol CrO₄ ²⁻ min⁻¹ mg protein⁻¹ for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203. Received 05 January 2000/ Accepted in revised form 27 May 2000     

Membrane potentials,electrolyte contents,cell pH,and some enzyme activities of fibroblasts

Summary The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was −10.2±0.20 mV (n=390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3′,5′-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10⁻⁵ to 10⁻⁴ M), hydrocortisone (10⁻⁷ to 10⁻⁶ M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na⁺, K⁺, and Cl⁻ concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [¹⁴C] dimethyloxazolidine-2, 4-dione, and³H₂O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activiities of Na⁺, K⁺-, HCO₃ ⁻-, and Ca⁺⁺, Mg⁺⁺-ATPases in these cultured cells were 19.0±2.1, 13.6±2.1, and 6.6±1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na⁺, K⁺, Cl⁻, and H⁺ are not distributed according to their electrochemical gradients across the cell membrane. Na⁺, Cl⁻, and H⁺ are actively transported out of the cells and K⁺ into the cells. This study was supported by Grant AM20935 from the NIAMDD, NIH, Bethesda, Maryland, and National Aeronautics & Space Administration NASA-Ames Grant NAG 2-108 and U.S. Department of Energy Contract DE-AC02-76-EV-00119. D. M. W. is the recipient of a Research Career Award (5-K6-NB-13838), NINCDS, NIH.     

Osmoregulation in juvenile and adult white sturgeon,Acipenser transmontanus

Synopsis Blood samples from cannulated young adult (2.5–15 kg) white sturgeon, acclimated to San Francisco Bay water (24 ppt) had plasma values of 248.8 ± 13.5 mOsm kg⁻¹ H₂O, [Na⁺] = 125 ± 8.0 mEq 1⁻¹, [K⁺] = 2.6 ± 0.8 mEq 1⁻¹ and [CL⁻] = 122 ± 3.0 mEq 1⁻¹. Freshwater acclimated sturgeon had an osmolality of 236 ± 7, [Na⁺] = 131.6 + 4.4, [K⁺] = 2.5 ± 0.7 and [CL⁻] = 110.6 ± 3.6. Freshwater acclimated fish gradually exposed to sea water (increase of 5 ppt h⁻¹) had higher plasma osmolalities than did the bay water acclimated fish. These young adult sturgeon are able to tolerate transfer from fresh water to sea water as well as gradual transfer from sea water to fresh water. Plasma electrolytes in transferred fish are regulated, but tend to differ from long term acclimated fish at the same salinities. There is a gradual increase in the upper salinity tolerance (abrupt transfer) of juvenile white sturgeon with weight: 5–10 ppt for 0.4–0.9 g fish, 10–15 ppt for 0.7–1.8 g fish, and 15 ppt for 4.9–50.0 g fish. The ability of juveniles to regulate plasma osmolality is limited. The young adult fish are able to tolerate higher salinities (35 ppt) than juvenile sturgeon but probably are also characterized by low activity of the necessary ion exchange mechanisms in the gills which permit rapid adjustment of blood electrolytes with graduate change in external salinity.     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

The ATPase activity of the <Emphasis Type="Italic">G2alt</Emphasis> gene encoding an aluminium tolerance protein from <Emphasis Type="Italic">Anoxybacillus gonensis</Emphasis> G2

The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range (25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT exhibited a low ATPase activity with K _m ⁻ and V _max ⁻ values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg²⁺ and Na⁺ ions, while Zn²⁺ and Al³⁺ stimulate the activity. Cd²⁺ and Ag⁺ reduced the activity and Li⁺, Cu²⁺, and Co²⁺ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily and it can be responsible for aluminium tolerance in A. gonensis G2.     

Growth and production of the bivalve <Emphasis Type="Italic">Macoma balthica</Emphasis> (Linnaeus, 1758) (Cardiida: Tellinidae) in the southeastern part of the Baltic Sea

The growth and production of the Baltic clam Macoma balthica in the southeastern part of the Baltic Sea were studied. The shell length of M. balthica reached 23.5 mm, the maximum age was 13+ years. The linear growth was described by the von Bertalanffy equation for shallow-water area (depths 9–40 m): L _τ = 23.99(1 − e ^{−0.1293(τ − (−0.9578))}), and for the deep-water area (41–81 m): L _τ = 20.61(1 − e ^{−0.1813(τ − (−0.5608))}). The annual production was lower (25.35 ± 1.72 kJ/m²) in the shallow-water area than in the deep-water area (71.23 ± 4.48 kJ/m²), with values of P _s /B ratio 0.44 and 0.38, respectively.     

Tree species and wood ash affect soil in Michigan’s Upper Peninsula

Tree species and wood ash application in plantations of short-rotation woody crops (SRWC) may have important effects on the soil productive capacity through their influence on soil organic matter (SOM) and exchangeable cations. An experiment was conducted to assess changes in soil C and N contents and pH within the 0–50 cm depth, and exchangeable cation (Ca²⁺, Mg²⁺, K⁺, and Na⁺) and extractable acidity concentrations within the 0–10 cm depth. The effects of different species (European larch [Larix decidua P. Mill.], aspen [Populus tremula L. × Populus tremuloides Michx.], and four poplar [Populus spp.] clones) and wood ash applications (0, 9, and 18 Mg ha⁻¹) on soil properties were evaluated, using a common garden experiment (N = 70 stands) over 7 years of management in Michigan’s Upper Peninsula. Soils were of the Onaway series (fine-loamy, mixed, active, frigid Inceptic Hapludalfs). The NM-6 poplar clone had the greatest soil C and N contents in almost all ash treatment levels. Soil C contents were 7.5, 19.4, and 10.7 Mg C ha⁻¹ greater under the NM-6 poplar than under larch in the ash-free, medium-, and high-level plots, respectively. Within the surface layer, ash application increased soil C and N contents (P < 0.05) through the addition of about 0.7 Mg C ha⁻¹ and 3 kg N ha⁻¹ with the 9 Mg ha⁻¹ ash application (twofold greater C and N amounts were added with the 18 Mg ha⁻¹ application). During a decadal time scale, tree species had no effects—except for K⁺—on the concentrations of the exchangeable cations, pH, and extractable acidity. In contrast, ash application increased soil pH and the concentration of Ca²⁺ (P < 0.05), from 5.2 ± 0.4 cmol_c kg⁻¹ (ash-free plots) to 8.6 ± 0.4 cmol_c kg⁻¹ (high-level ash plots), and tended to increase the concentration of Mg²⁺ (P < 0.1), while extractable acidity was reduced (P < 0.05) from 5.6 ± 0.2 cmol_c kg⁻¹ (ash-free plots) to 3.7 ± 0.2 cmol_c kg⁻¹ (high-level plots). Wood ash application, within certain limits, not only had a beneficial effect on soil properties important to the long-term productivity of fast-growing plantations but also enhanced long-term soil C sequestration.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Chemiluminescent in vitro estimation of the inhibitory constants of antioxidants ascorbic and uric acids in Fenton’s reaction in urine

The goal of this research was to measure in vitro the inhibitory constants of the antioxidants ascorbic and uric acid in urine, with lucigenin enhanced chemiluminescence (CL) in Fenton’s system. Maximum CL emission is registered in urine containing H₂O₂ (5·10⁻⁴ M), Fe²⁺ (5·10⁻⁵ M), EDTA (5·10⁻⁵ M), and chemical enhancer lucigenin (10⁻⁴ M) at pH 5.5 and 36°C. Ascorbic acid exhibits up to 4-fold stronger antioxidant effect than uric acid. The constants of antioxidant inhibition in urine were measured at concentrations 10⁻³ and 10⁻⁴ M: for ascorbic acid, 5.92 ± 0.04 and 24.05 ± 1.82 μmol·sec⁻¹; for uric acid, 1.60 ± 0.02 and 21.45 ± 0.97 μmol·sec⁻¹, respectively. Three phases of CL kinetics of urine are well observed: spontaneous CL (0–10 sec), fast flash of CL (10–50 sec), and latent period (50–300 sec). The antioxidant efficiency of ascorbic and uric acids in the final stage of catabolic processes in the body is discussed. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 8, pp. 1062–1065.     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

The impact of different chelating leaving groups on the substitution kinetics of mononuclear PtII(1,2- trans - R , R -diaminocyclohexane)(X–Y) complexes

A set of three oxaliplatin derivatives containing 1,2-trans-R,R-diaminocyclohexane (dach) as a spectator ligand and different chelating leaving groups X–Y, viz., [Pt(dach)(O,O-cyclobutane-1,1-dicarboxylate)], or Pt(dach)(CBDCA), [Pt(dach)(N,O-glycine)]⁺, or Pt(dach)(gly), and [Pt(dach)(N,S-methionine)]⁺, or Pt(dach)(l-Met), where l-Met is l-methionine, were synthesized and the crystal structure of Pt(dach)(gly) was determined by X-ray diffraction. The effect of the leaving group on the reactivity of the resulting Pt(II) complexes was studied for the nucleophiles thiourea, glutathione (GSH) and l-Met under pseudo-first-order conditions as a function of nucleophile concentration and temperature, using UV–vis spectrophotometric techniques. ¹H NMR spectroscopy was used to follow the substitution of the leaving group by guanosine 5′-monophosphate (5′-GMP²⁻) under second-order conditions. The rate constants indicate for all reactions a direct substitution of the X–Y chelate by the selected nucleophiles, thereby showing that the nature of the chelate, viz., O–O (CBDCA²⁻), N–O (glycine) or S–N (l-Met), respectively, plays an important role in the kinetic and mechanistic behavior of the Pt(II) complex. The k ₁ values for the reaction with thiourea, l-Met, GSH and 5′-GMP²⁻ were found to be as follows (10³ k ₁, 37.5 °C, M⁻¹ s⁻¹): Pt(dach)(CBDCA) 61 ± 2, 21.6 ± 0.1, 23 ± 1, 0.352 ± 0.002; Pt(dach)(gly) 82 ± 3, 6.2 ± 0.2, 37 ± 1, 1.77 ± 0.01; Pt(dach)(l-Met) (thiourea, GSH) 62 ± 2, 24 ± 1. The activation parameters for all reactions studied suggest an associative substitution mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

The metabolic responses and acid–base status after feeding, exhaustive exercise, and both feeding and exhaustive exercise in Chinese catfish (Silurus asotus Linnaeus)

Feeding and exhaustive exercise are known to elevate metabolism. However, acid–base status may be oppositely affected by the two processes. In this study, we first investigated the acid–base response of Chinese catfish to feeding (the meal size was about 8% of body mass) to test whether an alkaline tide (a metabolic alkalosis created by gastric HCl secretion after feeding) would occur. We then determined the combined effects of feeding and exhaustive exercise on excess post-exercise oxygen consumption and acid–base status to determine whether the alkaline tide induced by feeding protects against acid–base disturbance during exhaustive exercise and affects subsequent recovery. Arterial blood pH increased from 7.74 ± 0.02 before feeding to 7.88 ± 0.02 and plasma [HCO₃ ⁻]_pl increased from 5.42 ± 0.29 to 7.83 ± 0.37 mmol L⁻¹ 6 h after feeding, while feeding had no significant effect on P_\textCO2 P_{{{\text{CO}}_{2} }} . Exhaustive exercise led to a significant reduction in pH by 0.46 units and a reduction of [HCO₃ ⁻]_pl by ~3 mmol L⁻¹. Lactate concentrations in white muscle and plasma increased by 2.4 mmol L⁻¹ and 13.4 μmol g⁻¹, respectively. Fed fish had a higher pH and [HCO₃ ⁻]_pl than fasting fish at rest, and the reductions in pH (0.36 units) and [HCO₃ ⁻]_pl (~2 mmol L⁻¹) were thus lower after exhaustive exercise. However, the recovery of acid–base status and metabolites were similar in digesting and fasting fish. Overall, a significant alkaline tide was found in Chinese catfish after feeding. The alkaline tide elicited by feeding significantly prevented the decreases in pH and [HCO₃ ⁻]_pl immediately after exhaustive exercise, but recovery from exhaustive exercise was not affected by digestion.     

Production,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.