Metabolism of N-alkylated spermine analogues by polyamine and spermine oxidases

N-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus potentially limiting their utility. Here, we have studied the degradation of three different spermine analogues N,N′-bis-(3-ethylaminopropyl)butane-1,4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N′-(3-ethylaminopropyl)butane-1,4-diamine (BnEtSPM) and N,N′-bis-(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and related mono-alkylated derivatives as substrates of recombinant human polyamine oxidase (APAO) and spermine oxidase (SMO). APAO and SMO metabolized DESPM to EtSPD [K _m(APAO) = 10 μM, k _cat(APAO) = 1.1 s⁻¹ and K _m(SMO) = 28 μM, k _cat(SMO) = 0.8 s⁻¹, respectively], metabolized BnEtSPM to EtSPD [K _m(APAO) = 0.9 μM, k _cat(APAO) = 1.1 s⁻¹ and K _m(SMO) = 51 μM, k _cat(SMO) = 0.4 s⁻¹, respectively], and metabolized DBSPM to BnSPD [K _m(APAO) = 5.4 μM, k _cat(APAO) = 2.0 s⁻¹ and K _m(SMO) = 33 μM, k _cat(SMO) = 0.3 s⁻¹, respectively]. Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD [EtSPM K _m(APAO) = 16 μM, k _cat(APAO) = 1.5 s⁻¹; K _m(SMO) = 25 μM, k _cat(SMO) = 8.2 s⁻¹; BnSPM K _m(APAO) = 6.0 μM, k _cat(APAO) = 2.8 s⁻¹; K _m(SMO) = 19 μM, k _cat(SMO) = 0.8 s⁻¹, respectively]. Surprisingly, EtSPD [K _m(APAO) = 37 μM, k _cat(APAO) = 0.1 s⁻¹; K _m(SMO) = 48 μM, k _cat(SMO) = 0.05 s⁻¹] and BnSPD [K _m(APAO) = 2.5 μM, k _cat(APAO) = 3.5 s⁻¹; K _m(SMO) = 60 μM, k _cat(SMO) = 0.54 s⁻¹] were metabolized to SPD by both the oxidases. Furthermore, we studied the degradation of DESPM, BnEtSPM or DBSPM in the DU145 prostate carcinoma cell line. The same major metabolites EtSPD and/or BnSPD were detected both in the culture medium and intracellularly after 48 h of culture. Moreover, EtSPM and BnSPM were detected from cell samples. Present data shows that inducible SMO parallel with APAO could play an important role in polyamine based drug action, i.e. degradation of parent drug and its metabolites, having significant impact on efficiency of these drugs, and hence for the development of novel N-alkylated polyamine analogues.     

Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease

Chemical Exchange Saturation Transfer (CEST) is an MRI approach that can indirectly detect exchange broadened protons that are invisible in traditional NMR spectra. We modified the CEST pulse sequence for use on high-resolution spectrometers and developed a quantitative approach for measuring exchange rates based upon CEST spectra. This new methodology was applied to the rapidly exchanging Hδ1 and Hε2 protons of His57 in the catalytic triad of bovine chymotrypsinogen-A (bCT-A). CEST enabled observation of Hε2 at neutral pH values, and also allowed measurement of solvent exchange rates for His57-Hδ1 and His57-Hε2 across a wide pH range (3–10). Hδ1 exchange was only dependent upon the charge state of the His57 (k _ex,Im+ = 470 s⁻¹, k _ex,Im = 50 s⁻¹), while Hε2 exchange was found to be catalyzed by hydroxide ion and phosphate base ( k_{\textOH ^-} k_{{{\text{OH}}^{ - } }}  = 1.7 × 10¹⁰ M⁻¹ s⁻¹, k_{\textHPO4^{2 -}} k_{{{\text{HPO}}_{4}^{2 - } }}  = 1.7 × 10⁶ M⁻¹ s⁻¹), reflecting its greater exposure to solute catalysts. Concomitant with the disappearance of the Hε2 signal as the pH was increased above its pK _a, was the appearance of a novel signal (δ = 12 ppm), which we assigned to Hγ of the nearby Ser195 nucleophile, that is hydrogen bonded to Nε2 of neutral His57. The chemical shift of Hγ is about 7 ppm downfield from a typical hydroxyl proton, suggesting a highly polarized O–Hγ bond. The significant alkoxide character of Oγ indicates that Ser195 is preactivated for nucleophilic attack before substrate binding. CEST should be generally useful for mechanistic investigations of many enzymes with labile protons involved in active site chemistry.     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

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.     

Characterization of a recombinant endo-type alginate lyase (Alg7D) from <Emphasis Type="Italic">Saccharophagus degradans</Emphasis>

A gene, alg7D, from Saccharophagus degradans, coding for a putative alginate lyase belonging to the family of polysaccharide lyase-7, was overexpressed in Escherichia coli. The properties of the recombinant Alg7D were characterized. The enzyme endolytically depolymerized alginate by β-elimination into oligo-alginates with degrees of polymerization of 2–5. Its activity was maximal at 50°C and pH 7 and was slightly increased in the presence of Na⁺. The K _M , V _max , k _cat , and k _cat /K _M values were: 3 mg ml⁻¹, 6.2 U mg⁻¹, 1.9 × 10⁻² s⁻¹, and 6.3 × 10⁻³ mg⁻¹ ml s⁻¹, respectively.     

Finite element–based injury metrics for pulmonary contusion via concurrent model optimization

This study explores the relationship between impact severity and resulting pulmonary contusion (PC) for four impact conditions using a rat model of the injury. The force–deflection response from a Finite Element (FE) model of the lung was simultaneously matched to experimental data from distinct impacts via a genetic algorithm optimization. Sprague-Dawley rats underwent right-side thoracotomy prior to impact. Insults were applied directly to the lung via an instrumented piston. Five cohorts were tested: a sham group and four groups experiencing lung insults of varying degrees of severity. The values for impact velocity (V) and penetration depth (D) of the cohorts were Group 1, (V = 6.0 m · s⁻¹, D = 5.0 mm), Group 2, (V = 1.5 m · s⁻¹,D = 5.0 mm), Group 3, (V = 6 m · s⁻¹, D = 2.0 mm), and Group 4, (V = 1.5 m · s⁻¹, D = 2.0 mm). CT scans were acquired at 24 h, 48 h, and 1 week post-insult. Contusion volume was determined through segmentation. FE-based injury metrics for PC were determined at 24 h and 1 week post-impact, based on the observed volume of contusion and first principal strain. At 24 h post-impact, the volume of high radiopacity lung (HRL) was greatest for the severe impact group (mean HRL = 9.21 ± 4.89) and was significantly greater than all other cohorts but Group 3. The concurrent optimization matched simulated and observed impact energy within one standard deviation for Group 1 (energy = 3.88 ± 0.883 mJ, observed vs. 4.47 mJ, simulated) and Group 2 (energy = 1.46 ± 0.403 mJ, observed vs. 1.50 mJ, simulated) impacts. Statistically significant relationships between HRL and impact energy are presented. The FEA-based injury metrics at 24 h post-contusion are e_max·[(e)\dot]_max{\varepsilon_{\max}\cdot \dot {\varepsilon}_{\max}} exceeding 94.5 s⁻¹, ε _max exceeding 0.284 and [(e)\dot]_max{\dot{\varepsilon}_{\max}} exceeding 470 s⁻¹. Thresholds for injury to the lung still present at 1 week post-impact were also determined. They are e_max·[(e)\dot]_max{\varepsilon_{\max}\cdot \dot {\varepsilon}_{\max}} exceeding 149 s⁻¹, ε _max exceeding 0.343 and [(e)\dot]_max{\dot{\varepsilon}_{\max}} exceeding 573 s⁻¹. A mesh sensitivity study found that thresholds based on strain rate were more sensitive to changes to mesh density than the threshold based on strain only.     

The Relative Rates of Thiol–Thioester Exchange and Hydrolysis for Alkyl and Aryl Thioalkanoates in Water

This article reports rate constants for thiol–thioester exchange (k _ex), and for acid-mediated (k _a), base-mediated (k _b), and pH-independent (k _w) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate—model alkyl and aryl thioalkanoates, respectively—in water. Reactions such as thiol–thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k _a = 1.5 × 10⁻⁵ M⁻¹ s⁻¹, k _b = 1.6 × 10⁻¹ M⁻¹ s⁻¹, and k _w = 3.6 × 10⁻⁸ s⁻¹. At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol–thioester exchange between S-methyl thioacetate and 2-sulfonatoethanethiolate is k _ex = 1.7 M⁻¹ s⁻¹. At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions (pH, temperature, pK _a of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and rates of thiol–thioester exchange exceed those of hydrolysis by several orders of magnitude.     

Eco-physiological responses of nitrogen-fixing cyanobacteria to light

The eco-physiological responses of three nitrogen-fixing cyanobacteria (N-fixing cyanobacteria), Aphanizomenon gracile, Anabaena minderi, and Ana. torques-reginae, to light were assessed under nutrient saturation. The N-fixing cyanobacteria were isolated into monocultures from a natural bloom in a shallow colored lake and their growth irradiance parameters and pigment composition were assessed. The different ecological traits related to light use (μ_max, α, I _k) suggest that these N-fixing cyanobacteria are well adapted to low light conditions at sufficient nutrients, yet interspecific differences were observed. Aphanizomenon gracile and Anabaena minderi had high relative growth rates at low irradiances (ca. 70% of those in high light), low half saturation constant for light-limited growth (I _k < 9.09 μmol photon m⁻² s⁻¹) and high efficiency (α < 0.11 day⁻¹ μmol photon⁻¹ m² s). Conversely, Ana. torques-reginae showed poorer light competitiveness: low relative growth rates at low irradiances (ca. 40% of those in high light), low α (0.009 day⁻¹ μmol photon⁻¹ m² s) and higher I _k (35.5 μmol photon m⁻² s⁻¹). Final densities in Aphanizomenon gracile and Anabaena minderi reached bloom densities at irradiances above 30 μmol photon m⁻² s⁻¹ with different hierarchy depending on irradiance, whereas Ana. torques-reginae never achieved bloom densities. All species had very low densities at irradiances ≤17 μmol photon m⁻² s⁻¹, thus no N-fixing blooms would be expected at these irradiances. Also, under prolonged darkness and at lowest irradiance (0 and 3 μmol photon m⁻² s⁻¹) akinetes were degraded, suggesting that in ecosystems with permanently dark sediments, the prevalence of N-fixing cyanobacteria should not be favored. All species displayed peaks of phycocyanin, but no phycoeritrin, probably due to the prevailing red light in the ecosystem from which they were isolated.     

Preliminary investigation of Okhotsk Sea ice algae; taxonomic composition and photosynthetic activity

Okhotsk Sea pack ice from Shiretoko in northern Hokkaido, sampled in March 2007, contained microalgal communities dominated by the centric diatoms Thalassiosira nordenskioeldi and T. punctigera. Domination by this genus is very unusual in sea ice. Communities from nearby fast ice at Saroma-ko lagoon were dominated by Detonula conferavea and Odontella aurita. Average microalgal biomass of the Okhotsk Sea pack ice (surface and bottom) was 1.59 ± 1.09 μg chla l⁻¹ and for fast ice (bottom only) at nearby Saroma-ko lagoon, 16.5 ± 3.2 μg l⁻¹ (=31.1 ± 5.0 mg chla m⁻²). Maximum quantum yield of the Shiretoko pack ice algal communities was 0.618 ± 0.056 with species-specific data ranging between 0.211 and 0.653. These community values are amongst the highest recorded for sea ice algae. Rapid light curves (RLC) on individual cells indicated maximum relative electron transfer rates (relETR) between 20.8 and 60.6, photosynthetic efficiency values (α) between 0.31 and 0.93 and onset of saturation values (E _k) between 33 and 91 μmol photons m⁻² s⁻¹. These data imply that the pack ice algal community at Shiretoko was healthy and actively photosynthesising. Maximum quantum yield of the Saroma-ko fast ice community was 0.401 ± 0.086, with values for different species between 0.361 and 0.560. RLC data from individual Saroma-ko fast ice algal cells indicated relETR between 55.3 and 60.6, α values between 0.609 and 0.816 and E _k values between 74 and 91 μmol photons m⁻² s⁻¹ which are consistent with measurements in previous years.     

Gallium uptake by transferrin and interaction with receptor 1

The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate in about 50 s to yield an intermediate complex with an equilibrium constant K ₁ = (3.9 ± 1.2) × 10⁻², a direct second-order rate constant k ₁ = 425 ± 50 M⁻¹ s⁻¹ and a reverse second-order rate constant k ₋₁ = (1.1 ± 3) × 10⁴ M⁻¹ s⁻¹. The intermediate complex loses a single proton with proton dissociation constant K _1a = 80 ± 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant K _d = 1.10 ± 0.12 μM and a second-order rate constant k _d = (1.15 ± 0.3) × 10¹⁰ M⁻¹ s⁻¹. This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition pathway is discussed.     

Cloning and characterization of a ribitol dehydrogenase from Zymomonas mobilis

Ribitol dehydrogenase (RDH) catalyzes the conversion of ribitol to d-ribulose. A novel RDH gene was cloned from Zymomonas mobilis subsp. mobilis ZM4 and overexpressed in Escherichia coli BL21(DE3). DNA sequence analysis revealed an open reading frame of 795 bp, capable of encoding a polypeptide of 266 amino acid residues with a calculated molecular mass of 28,426 Da. The gene was overexpressed in E. coli BL21(DE3) and the protein was purified as an active soluble form using glutathione S-transferase affinity chromatography. The molecular mass of the purified enzyme was estimated to be ∼28 kDa by sodium dodecyl sulfate-polyacrylamide gel and ∼58 KDa with gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 9.5 and 65°C, respectively. Unlike previously characterized RDHs, Z. mobilis RDH (ZmRDH) showed an unusual dual coenzyme specificity, with a k _cat of 4.83 s⁻¹ for NADH (k _cat/K _m = 27.3 s⁻¹ mM⁻¹) and k _cat of 2.79 s⁻¹ for NADPH (k _cat/K _m = 10.8 s⁻¹ mM⁻¹). Homology modeling and docking studies of NAD⁺ and NADP⁺ into the active site of ZmRDH shed light on the dual coenzyme specificity of ZmRDH.     

Effects of trans-pyridine ligands on the interactions of RuII and RuIII ammine complexes N7-coordinated to purine nucleosides and DNA

 DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, 4-bnpy) is highly selective for G⁷ with K _G=1.1×10⁴ to 2.8×10⁴, with the more hydrophobic Pyr ligands exhibiting slightly higher binding. A strong dependence on ionic strength indicates that ion-pairing with DNA occurs prior to binding. At μ=0.05, d[Ru^II-DNA]/dt=k[Ru^II][DNA], where k=0.17–0.21 M^–1s^–1 with the various Pyr ligands. The air oxidation of [(py)(NH₃)₄Ru^II] _n -DNA to [(py)(NH₃)₄Ru^III] _n -DNA at pH 6 occurs with a pseudo-first-order rate constant of k _obs=5.6×10^–4s^–1 at μ=0.1, T=25  °C. Strand cleavage of plasmid DNA appears to occur by both Fenton/Haber-Weiss chemistry and by base-catalyzed routes, some of which are independent of oxygen. Base-catalyzed cleavage is more efficient than O₂ activation at neutral pH and involves the disproportionation of covalently bound Ru^III and, in the presence of O₂, Ru-facilitated autoxidation to 8-oxoguanine. Disproportionation of [py(NH₃)₄Ru^III] _n -DNA occurs according to the rate law: d[Ru^II–G_DNA]/dt=k ₀[Ru^III–G_DNA]+k ₁[Ru^III–G_DNA][OH^–], where k ₀=5.4×10^–4s^–1 and k ₁=8.8 M^–1s^–1 at 25  °C, μ=0.1. The appearance of [(Gua)(py)(NH₃)₄Ru^III] under argon, which occurs according to the rate law: d[Ru^III–G]/dt=k ₀[Ru^III–G_DNA]+k ₁[OH^–][Ru^III–G_DNA] (k ₀=5.74×10^–5 s^–1, k ₁=1.93×10^–2M^–1s^–1 at T=25  °C, μ=0.1), is consistent with lysis of the N-glycosidic bond by Ru^IV-induced general acid hydrolysis. In air, the ratio of [Ru-8-OG]/[Ru-G] and their net rates of appearance are 1.7 at pH 11, 25  °C. Small amounts of phosphate glycolate indicate a minor oxidative pathway involving C4′ of the sugar. In air, a dynamic steady-state system arises in which reduction of Ru^IV produces additional Ru^II. Received: 11 November 1998 / Accepted: 3 March 1999     

Characterization of a thermostable endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from <Emphasis Type="Italic">Caldicellulorsiruptor saccharolyticus</Emphasis>

A recombinant putative glycoside hydrolase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 12 U mg⁻¹ by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band upon SDS-PAGE. The native enzyme is a dimer with a molecular mass of 112 kDa as determined by gel filtration. The enzyme exhibited its highest activity when debranched arabinan (1,5-α-l-arabinan) was used as the substrate, demonstrating that the enzyme was an endo-1,5-α-l-arabinanase. The K _m, k _cat, and k _cat/K _m values were 18 mg ml⁻¹, 50 s⁻¹, and a 2.8 mg ml⁻¹ s⁻¹, respectively. Maximum enzyme activity was at pH 6.5 and 75°C. The half-lives of the enzyme at 65, 70 and 75°C were 2440, 254 and 93 h, respectively, indicating that it is the most thermostable of the known endo-1,5-α-l-arabinanases.     

Metallopeptide-promoted inactivation of angiotensin-converting enzyme and endothelin-converting enzyme 1: toward dual-action therapeutics

A series of metallopeptides based on the amino terminal copper/nickel (ATCUN) binding motif have been evaluated as classical inhibitors and catalytic inactivators of both rabbit and human angiotensin-converting enzyme (hACE), and human endothelin-converting enzyme 1 (hECE-1). The cobalt complex [KGHK–Co(NH₃)₂]²⁺, where KGHK is lysylglycylhistidyllysine, displayed similar K _I and IC₅₀ values to those found for [KGHK–Cu]⁺, in spite of the enhanced charge, and so either the influence of charge is offset by the steric influence of the axially coordinated ammine ligands, or binding is dominated by contributions from the amino acid side chains, especially the C-terminal lysine that mimics the binding pattern observed for lisinopril. Moreover, the inhibition observed for [KGHK–Co(NH₃)₂]²⁺ contrasts with the activation of hACE by Co²⁺(aq), reflecting the stimulation of enzyme activity following replacement of the catalytic zinc cofactor by cobalt ion at each of the two active sites. Quantitative analysis of the dose-dependent stimulation of activity by Co²⁺(aq) yielded apparent affinities of 1.3 ± 0.2 and 56 ± 8 μM for the two sites in the presence of saturating Zn²⁺ (10 μM). Catalytic inactivation of hACE by [KGHK–Cu] ⁺ at subsaturating concentrations had previously been characterized, with k _obs = 2.9 ± 0.5 × 10⁻² min⁻¹. Under similar conditions, the same complex is found to catalytically inactivate hECE-1, with k _obs = 2.12 ± 0.16 × 10⁻² min⁻¹, demonstrating the potential for dual-action activity against two key drug targets in cardiovascular disease. Irreversible inactivation of a drug target represents a novel mechanism of drug action that complements existing classical inhibitor strategies that underlie current drug discovery efforts.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Energetics of swimming at maximal speeds in humans

The energy cost per unit of distance (C _s, kilojoules per metre) of the front-crawl, back, breast and butterfly strokes was assessed in 20 elite swimmers. At sub-maximal speeds (v), C _s was measured dividing steady-state oxygen consumption (V˙O₂) by the speed (v, metres per second). At supra-maximal v, C _s was calculated by dividing the total metabolic energy (E, kilojoules) spent in covering 45.7, 91.4 and 182.9 m by the distance. E was obtained as: E = E _an+V˙O_2max t _p−V˙O_2max(1−e^{−( t p/)}), where E _an was the amount of energy (kilojoules) derived from anaerobic sources, V˙O_2max litres per second was the maximal oxygen uptake, α (=20.9 kJ · l O₂ ⁻¹) was the energy equivalent of O₂, τ (24 s) was the time constant assumed for the attainment of V˙O_2max at muscle level at the onset of exercise, and t _p (seconds) was the performance time. The lactic acid component was assumed to increase exponentially with t _p to an asymptotic value of 0.418 kJ · kg⁻¹ of body mass for t _p ≥ 120 s. The lactic acid component of E _an was obtained from the net increase of lactate concentration after exercise (Δ[La]_b) assuming that, when Δ[La]_b = 1 mmol · l⁻¹ the net amount of metabolic energy released by lactate formation was 0.069 kJ · kg⁻¹. Over the entire range of v, front crawl was the least costly stroke. For example at 1 m · s⁻¹, C _s amounted, on average, to 0.70, 0.84, 0.82 and 0.124 kJ · m⁻¹ in front crawl, backstroke, butterfly and breaststroke, respectively; at 1.5 m · s⁻¹, C _s was 1.23, 1.47, 1.55 and 1.87 kJ · m⁻¹ in the four strokes, respectively. The C _s was a continuous function of the speed in all of the four strokes. It increased exponentially in crawl and backstroke, whereas in butterfly C _s attained a minimum at the two lowest v to increase exponentially at higher v. The C _s in breaststroke was a linear function of the v, probably because of the considerable amount of energy spent in this stroke for accelerating the body during the pushing phase so as to compensate for the loss of v occurring in the non-propulsive phase. Accepted: 14 April 1998     

Purification and characterisation of lignin peroxidase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> MTCC-137

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K _m, k _cat and k _cat/K _m values of the enzyme using veratryl alcohol and H₂O₂ as the substrate were 61 M, 2.13 s⁻¹, 3.5 × 10⁴ M⁻¹s⁻¹ and 71 M, 2.13 s⁻¹, 3.0 × 10⁴ M⁻¹ s⁻¹ respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25°C.     

Quadrupedal locomotor performance in two species of arboreal squirrels: predicting energy savings of gliding

Gliding allows mammals to exploit canopy habitats of old-growth forests possibly as a means to save energy. To assess costs of quadrupedal locomotion for a gliding arboreal mammal, we used open-flow respirometry and a variable-speed treadmill to measure oxygen consumption and to calculate cost of transport, excess exercise oxygen consumption, and excess post-exercise oxygen consumption for nine northern flying squirrels (Glaucomys sabrinus) and four fox squirrels (Sciurus niger). Our results indicate that oxygen consumption during exercise by flying squirrels was 1.26–1.65 times higher than predicted based on body mass, and exponentially increased with velocity (from 0.84 ± 0.03 ml O₂ kg⁻¹ s⁻¹ at 0.40 m s⁻¹ to 1.55 ± 0.03 ml O₂ kg⁻¹ s⁻¹ at 0.67 m s⁻¹). Also, cost of transport in flying squirrels increased with velocity, although excess exercise oxygen consumption and excess post-exercise oxygen consumption did not. In contrast, oxygen consumption during exercise for fox squirrels was similar to predicted, varying from 0.51 (±0.02) ml O₂ kg⁻¹ s⁻¹ at 0.63 m s⁻¹ to 0.54 (±0.03) ml O₂ kg⁻¹ s⁻¹ at 1.25 m s⁻¹. In addition, the cost of transport for fox squirrels decreased with velocity, while excess exercise oxygen consumption and excess post-exercise oxygen consumption did not. Collectively, these observations suggest that unlike fox squirrels, flying squirrels are poorly adapted to prolonged bouts of quadrupedal locomotion. The evolution of skeletal adaptations to climbing, leaping, and landing and the development of a gliding membrane likely has increased the cost of quadrupedal locomotion by >50% while resulting in energy savings during gliding and reduction in travel time between foraging patches.     

Role of the left aortic arch and blood flows in embryonic American alligator (<Emphasis Type="Italic">Alligator mississippiensis</Emphasis>)

All embryonic and fetal amniotes possess a ductus(i) arteriosus(i) that allows blood to bypass the pulmonary circulation and the non-functional lungs. The central hemodynamic of embryonic reptiles are unique, given the additional systemic aorta that allows pulmonary circulatory bypass, the left aorta (LAo). The LAo exits in the right ventricle or ‘pulmonary side’ of reptilian hearts in both embryos and adults, but its functional significance in ovo is unknown. This study investigated the role of the LAo in embryonic American alligators by surgically occluding the LAo and measuring oxygen consumption and, in addition, measured hemodynamic responses to hypoxia in embryonic alligators. We measured systemic cardiac output and primary chorioallantoic membrane (CAM) artery blood flow for normoxic and hypoxic-incubated (10% O₂) American alligator embryos (Alligator mississippiensis). Chronic blood flow (1–124 h) in the primary CAM artery for hypoxic-incubated embryos (92 ± 26 ml min⁻¹ kg⁻¹) was elevated when compared with normoxic-incubated embryos (29 ± 14 ml min⁻¹ kg⁻¹, N = 6; P = 0.039). For hypoxic-incubated embryos, acute LAo blood flow (49.6 ± 24.4 ml min⁻¹ kg⁻¹) was equivalent to the combined flow of the three systemic great vessels that arise from the left ventricle, the right aorta, common carotid and subclavian arteries (43.6 ± 21.5 ml min⁻¹ kg⁻¹, N = 5). Similarly, for normoxic-incubated embryos, LAo blood flow (27.3 ± 6.6 ml min⁻¹ kg⁻¹) did not statistically differ from the other three vessels (18.4 ± 4.9 ml min⁻¹ kg⁻¹, N = 5). This study contains the first direct test of LAo function and the first measurements of blood flow in an embryonic reptile. These data support the hypotheses that embryonic alligators utilize the LAo to divert a significant amount of right ventricular blood into the systemic circulation, and that CAM blood flow increases following chronic hypoxic conditions. However, surgical occlusion of the LAo did not affect egg [(V)\dot]_\textO2, \dot{V}_{{\text{O}}_{2}}, supporting the hypothesis that the LAo of reptiles is not critical to maintain in ovo oxygen consumption.     

Biokinetic parameters and behavior of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> during anaerobic growth

The biokinetics of glucose metabolism were evaluated in Aeromonas hydrophila during growth in an anaerobic biosystem. After approx 34 h growth, A. hydrophila metabolized 5,000 mg glucose l⁻¹ into the end-products ethanol, acetate, succinate and formate. The maximum growth rate, μ _m, half saturation coefficients, K _s, microbial yield coefficient, Y, cell mass decay rate coefficient, k _d, and substrate inhibition coefficient, K _si were 0.25 ± 0.03 h⁻¹, 118 ± 31 mg glucose l⁻¹, 0.12 μg DNA mg glucose⁻¹, 0.01 h⁻¹, and 3,108 ± 1,152 mg glucose l⁻¹, respectively. These data were used to predict the performance of a continuous growth system with an influent glucose concentration of 5,000 mg l⁻¹. Results of the analysis suggest that A. hydrophila will metabolize glucose at greater than 95% efficiency when hydraulic retention times (HRTs) exceed 7 h, whereas the culture is at risk of washing out at an HRT of 6.7 h.