Fast repair of purine deoxynucleotide radical cations by rutin and quercetin

Repair effects of rutin and quercetin on purine deoxynucleotide radical cations were studied using pulse radiolysis technique. On electron pulse irradiation of N2 saturated deoxynucleotide aqueous solution containing 20 mmol/L K2S2O8, 200 mmol/L f-BuOH and rutin or quercetin, the transient absorption spectra of the deoxynucleotide radical cations decayed quickly. At the same time, the spectra of flavonoid phenoxyl radicals formed within several dozen microseconds. The results indicated that deoxynucleotide radical cations can be repaired by flavonoids. The rate constants of the repair reactions were 3.8 × 108-4.4×108 mol-1 · L · s-1 and 1.3×108-1.8×108 mol-1 · L · s-1 for dAMP and dGMP radical cations, respectively.     

Reaction of hydroxyl radical with phenylpropanoid glycoside and its derivatives by pulse radiolysis

The reaction of hydroxyl radical with 1 phenylpropanoid glycoside (PPG), cistanoside C, and its 3 derivatives: 1-0-β-D-2-(p-hydroxyphenyl)-ethanyl-glucose, 6-O-(E)-femloyl-glucose and 6-O-(E)-p-hydroxy-cinnarnoylglucose isolated from folk medicinal herbs was investigated by pulse radiolysis technique respectively. The reaction rate constants were determined by analysis of built-up trace of absorption at λ_max of specific transient absorption spectra of PPG and its derivatives upon attacking · OH. All four compounds react with · OH at close to diffusion controlled rate (1. 03 × 10⁹-19.139 × 10⁹ L · mol⁻¹ · s⁻¹), suggesting that they are effective · OH scavengers. The results demonstrated that the numbers of phenolic hydroxyl groups of PPG and its derivatives are directly related to their scavenging activities. By comparing the reaction rates of · OH with 1-O-β-D-2-(p-hydroxyphenyl)-ethanyl-glucose, 6-O-(E)-feruloyl-glucose or 6-O-(E)-p-hydroxy-cinnomoyl-glucose, it is evident that the phenylethyl group is more impofiant than phenylacryloyl group for scavenging · OH.     

Reaction of hydroxyl radical with phenylpropanoid glycoside and its derivatives by pulse radiolysis

The reaction of hydroxyl radical with 1 phenylpropanoid glycoside ( PPG), cistanoside C, and its 3 derivatives: 1-O-β-D-2-(p-hydroxyphenyl)-ethanyl-glucose, 6-O-(E)-feruloyl-glucose and 6-O-(E)-p-hydroxy-cinnamoylglucose isolated from folk medicinal herbs was investigated by pulse radiolysis technique respectively. The reaction rate constants were determined by analysis of built-up trace of absorption at λ_(max) of specific transient absorption spectra of PPG and its derivatives upon attacking·OH. All four compounds react with·OH at close to diffusion controlled rate (1.03×10~9—19.139×10~9 L·mol~(-1)·s~(-1)), suggesting that they are effective·OH scavengers. The results demonstrated that the numbers of phenolic hydroxyl groups of PPG and its derivatives are directly related to their scavenging activities. By comparing the reaction rates of·OH with 1-O-β-D-2-(p-hydroxyphenyl)-ethanyl-glucose, 6-O-(E)-feruloyl-glueose or 6-O-(E)-p-hydroxy-cinnomoyl-glucose, it is evident that the phenylethyl g     

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.     

An Overview of DNA and RNA Bindings to Antioxidant Flavonoids

In this report we are examining how the antioxidant flavonoids can prevent DNA damage and what mechanism of action is involved in the process. Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. We study the interactions of quercetin (que), kaempferol (kae), and delphinidin (del) with DNA and transfer RNA in aqueous solution at physiological conditions, using constant DNA or RNA concentration 6.25 mmol (phosphate) and various pigment/polynucleotide(phosphate) ratios of 1/65 to 1 (DNA) and 1/48 to 1/8 (tRNA). The structural analysis showed quercetin, kaempferol, and delphinidin intercalate DNA and RNA duplexes with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants for DNA adducts K _que = 7.25 (±0.65) × 10⁴ M⁻¹, K _kae = 3.60 (±0.33) × 10⁴ M^−1, and K _del = 1.66 (±0.25) × 10⁴ M⁻¹ and for tRNA adducts K _que = 4.80 (±0.50) × 10⁴ M⁻¹, K _kae = 4.65 (±0.45) × 10⁴ M^−1, and K _del = 9.47 (±0.70) × 10⁴ M⁻¹. The stability of adduct formation is in the order of del>que>kae for tRNA and que>kae>del for DNA. Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial B to A-DNA transition occurs at high drug concentration, while tRNA remains in A-family structure. The antioxidant activity of flavonoids changes in order delphinidin>quercetin>kaempferol. The results show intercalated flavonoids can make them strong antioxidants to protect DNA from harmful free radical reactions.     

Characteristics of net ecosystem carbon dioxide exchange (NEE) from August to October of Alpine meadow on the Tibetan Plateau, China

The Alpine meadow is one of the vegetation types widely distributed on the Tibetan Plateau in China with an area of about 1.2 million square kilometers. The Damxung rangeland station, located in the hinterland of the Tibetan Plateau, is covered with an typical vegetation. The continuous carbon flux data (from August to middle October, 2003) measured with the open-path eddy covariance system was used to analyze the diurnal variation pattern of net ecosystem carbon dioxide exchange (NEE) and its relationship with the environmental factors, such as photosynthetically active radiation (PAR), precipitation, and temperature. Results showed that NEE presented obvious diurnal variation pattern with single-peak of diurnal maximum carbon assimilation at 11: 00–12: 00 (local time) with an average of −0.268 mg CO₂·m⁻²·s⁻¹, i.e., −6.08 μmol CO₂·m⁻²·s⁻¹. During the daytime, NEE fitted fairly well with PAR in a rectangular hyperbola function with the apparent quantum yield (0.020 3 μmol CO₂ μmol⁻¹ PAR) and maximum ecosystem assimilation (9.741 1 μmol CO₂·m⁻²·s⁻¹). During the night-time, NEE showed a good exponential relation with the soil temperature at 5 cm depth. __________ Translated from Acta Ecologica Sinica 2005, 25(8): 1948–1952 [译自: 生态学报, 2005, 25(8): 1948–1952]     

Fast repair of dAMP hydroxyl radical adduct by verbascoside via electron transfer

DNA damaged by oxygen radicals has been implicated as a causative event in a number of degenerative diseases, including cancer and aging. So it is very impotant to look for ways in which either oxygen radicals are scavenged prior to DNA damage or damaged DNA is repaired to supplement the cells' inadequate repair capacity. The repair activity and its mechanism of verbaseoside, isolated from Pedicularis species, towards dAMP-OH·was studied with pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mmol/L dAMP aqueous solution containing verbascoside, the transient absorption spectrum of the hydroxyl adduct of dAMP decayed with the formation of that of the phenoxyl radical of verbascoside well under 100 microseconds after electron pulse irradiation. The result indicated that dAMP hydroxyl adducts can be repaired by verbascoside. The rate constants of the repair reaction was deduced to be 5.9×10~8 dm~3·mol~(-1)·s~(-1). A deeper understanding of this new repair mechanism will undo     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Through-flow of water in leaves of a submerged plant is influenced by the apical opening

Submerged aquatic higher plants maintain acropetal water transport to the young leaves in active growth to satisfy their demand for nutrients and hormones derived from the roots. We here present the first measurements of hydraulic properties for a submerged plant, the monocotyledon Sparganium emersum Rehman. The hydraulic conductance per unit length, K_h, was measured in leaf segments without the leaf tip and shown to be greater in old, fully developed leaves (1.5 · 10⁻¹⁰ · m⁴ · MPa⁻¹ · s⁻¹) than in young leaves (1.0 · 10⁻¹⁰ · m⁴ · MPa⁻¹ · s⁻¹). In leaves with intact leaf tips, however, K_h was significantly greater in the youngest leaves, which suggests that the leaf tip with the hydathode influences resistance and thus flow. Microscopy confirmed that the hydathodal area, which is an apical opening, undergoes structural changes with leaf age; a matrix of microorganisms develops in the older leaves and probably restricts water flow by clogging the hydathodes. The leaf specific conductivity expressing transport capacity relative to the leaf area supplied, of S. emersum (0.1 · 10⁻⁸ to 9 · 10⁻⁸ · m² MPa⁻¹· s⁻¹) was within the same range as for various species of terrestrial ferns, vines and trees. This finding does not support the traditional concept of functionally reduced vascular transport in Received: 15 July 1996 / Accepted: 30 November 1996     

Laser flash photolysis study on antioxidant properties of hydroxycinnamic acid derivatives

The antioxidant properties of hydroxycinnamic acid derivatives (HCA) were studied by laser flash photolysis. The transient species with maximum absorption at 360 nm were assigned to the phenoxyl radical of HCA. The SO₄^•− induced oxidation of HCA was also investigated. It was shown that the interaction of SO₄^•− with HCA resulted in the formation of HCA phenoxyl radicals with rate constants of 2.0–3.9×10⁹ M⁻¹ s⁻¹. The reactions of HCA with triplet state of benzophenone were analyzed and quenching rate constants of 4.3–7.8×10⁹ M⁻¹ s⁻¹ were determined.     

Ab initio studies on the decomposition kinetics of CF<Subscript>3</Subscript>OCF<Subscript>2</Subscript>O radical

The present study deals with the decomposition of CF₃OCF₂O radical formed from a hydrofluoroether, CF₃OCHF₂ (HFE-125), in the atmosphere. The study is performed using ab initio quantum mechanical methods. Two plausible pathways of decomposition of the titled species have been considered, one involving C-O bond scission and the other occurring via F atom elimination. The geometries of the reactant, products and transition states involved in the decomposition pathways are optimized and characterized at DFT (B3LYP) level of theory using 6-311G(d,p) basis set. Single point energy calculations have been performed at G2M(CC,MP2) level of theory. Out of the two prominent decomposition channels considered, the C-O bond scission is found to be dominant involving a barrier height of 15.3 kcal mol⁻¹ whereas the F-elimination path proceeds with a barrier of 26.1 kcal mol⁻¹. The thermal rate constants for the above two decomposition pathways are evaluated using canonical transition state theory (CTST) and these are found to be 1.78 × 10⁶ s⁻¹ and 2.83 × 10⁻⁷ s⁻¹ for C-O bond scission and F-elimination respectively at 298 K and 1 atm pressure. Transition states are searched on the potential energy surfaces involved during the decomposition channels and each of the transition states is characterized. The existence of transition states on the corresponding potential energy surface is ascertained by performing intrinsic reaction coordinate (IRC) calculation.     

Ab initio studies on the reactivity of the CF3OCH2O radical: Thermal decomposition vs. reaction with O2

Hydrofluoroethers are being considered as potential candidates for third generation refrigerants. The present investigation involves the ab initio quantum mechanical study of the decomposition mechanism of CF₃OCH₂O radical formed from a hydrofluoroether, CF₃OCH₃ (HFE-143a) in the atmosphere. The geometries of the reactant, products and transition states involved in the decomposition pathways are optimized and characterized at the DFT (B3LYP) level of theory using 6-311G(d,p) basis set. Energy calculations have been performed at the G2(MP2) and G2M(CC,MP2) level of theory. Two prominent decomposition channels, C-O bond scission and reaction with atmospheric O₂ have been considered for detailed investigation. Studies performed at the G2(MP2) level reveals that the decomposition channel involving C-O bond scission occurs with a barrier height of 23.8 kcal mol⁻¹ whereas the oxidative pathway occurring with O₂ proceeds with an energy barrier of 7.2 kcal mol⁻¹. On the other hand the corresponding values at G2M(CC,MP2) are 24.5 and 5.9 kcal mol⁻¹ respectively. Using canonical transition state theory (CTST) rate constants for the two pathways considered are calculated at 298 K and 1 atm pressure and found to be 5.9 × 10⁻⁶ s⁻¹ and 2.3 × 10⁻⁵ s⁻¹ respectively. The present study concludes that reaction with O₂ is the dominant path for the consumption of CF₃OCH₂O in the atmosphere. Transition states are searched and characterized on the potential energy surfaces involved in both of the reaction channels. The existence of transition state on the corresponding potential energy surface is ascertained by performing intrinsic reaction coordinate (IRC) calculation.     

Growth and net photosynthetic rates of <Emphasis Type="Italic">Hosta</Emphasis> ‘blue vision’ during acclimatization in bright,natural light with CO<Subscript>2</Subscript> enrichment

Summary Hosta ‘Blue Vision’, a shade-adapted perennial, was successfully acclimatized in high, natural light conditions in the research Acclimatron^TM at Clemson University, Clemson, SC during the summer of 2000. The supplemental CO₂ levels achieved during acclimatization were 710±113, 2396±121, and 5641±119 μmol mol⁻¹, approximately 2×, 6×, and 15× ambient CO₂. Plants were maintained in H₂O-saturated atmospheres and protected from temperature increases associated with high light intensity. In the 5 wk following ex vitro transfer, plantlet roots grew at the 2× CO₂ level, but shoot biomass was unaffected. Results for the 6× and 15× CO₂ levels were comparable and provided the best plantlet growth. The “doubling time’ that is characteristic of exponential growth was 10.8 and 9.8 d for root and shoot dry weights, respectively. There was no indication of light saturation of net photosynthetic rate (NPR) over the photosynthetic photon flux density (PPFD) range of 100–1200 μmolm⁻²s⁻¹ experienced during this study. An interaction between CO₂ and light intensity levels was detected for NPR of Hosta ‘Blue Vision’ with CO₂ saturation occurring at approximately 2800 μmol mol⁻¹. regardless of light level. Furthermore, at the optimal CO₂ level, NPR increased quadratically as light intensity increased, and NPR was greatest at the maximum light intensity (PPFD: 1200 μmol m⁻²s⁻¹).     

The function of water channels in Chara: The temperature dependence of water and solute flows provides evidence for composite membrane transport and for a slippage of small organic solutes across water channels

Using the cell pressure probe, the effects of temperature on hydraulic conductivity (Lp; osmotic water permeability), solute permeability (permeability coefficient, P_s), and reflection coefficients (σ_s) were measured on internodes of Chara corallina, Klein ex Willd., em R.D.W.. For the first time, complete sets of transport coefficients were obtained in the range between 10 and 35 °C which provided evidence about pathways of water and solutes as they move across the plasma membrane (water channel and bilayer arrays). Test solutes used to check for the selectivity of water channels were monohydric alcohols of different molecular size and shape (ethanol, n-propanol, iso-propanol, and tert-butanol) and heavy water (HDO). Within the limits of accuracy, Q₁₀ values for Lp and for the diffusive water permeability (P_d) were identical (Q₁₀ for Lp = 1.29 ± 0.17 (± SD; n = 15 cells) and Q₁₀ for P_d = 1.25 ± 0.16 (n = 5 cells)). The Q₁₀ values were equivalent to activation energies of E_a = 16.8 ± 6.4 and 16.6 ± 10.0 kJ · mol⁻¹, respectively, which is similar to that of self-diffusion or of viscous flow of water. The Q₁₀ values and activation energies for P_s of the alcohols were significantly larger (ethanol: Q₁₀ = 1.68 ± 0.16, E_a = 37.1 ± 5.9 kJ · mol⁻¹; n-propanol: Q₁₀ =  1.75 ± 0.40, E_a = 43.1 ± 15.3 kJ · mol⁻¹; iso-propanol: Q₁₀ = 2.12 ± 0.42, E_a =  52.2 ± 14.6 kJ · mol⁻¹; tert-butanol: Q₁₀ = 2.13 ± 0.56, E_a = 51.6 ± 17.1 kJ · mol⁻¹; ±SD; n = 5 to 6 cells). Effects of temperature on reflection coefficients were most pronounced. With increasing temperature, σ_s values of the alcohols decreased and those of HDO increased. The data indicate that water and solutes use different pathways when crossing the membrane. Ordinary and isotopic water use water channels and the other test solutes use the bilayer array (composite transport model of membrane). Changes in σ_s values with temperature were found to be a sensitive measure for the open/closed state of water channels. The decrease of σ_s with temperature was theoretically predicted from the temperature dependence of P_s and Lp. Differences between predicted and measured values of σ_s allowed estimation of the bypass flow (slippage) of solutes through water channels which did not completely exclude test solutes. The permeability of channels depended on the structure and size of test solutes. It is concluded that water channels are much less selective than is usually thought. Since water channels represent single-file or no-pass pores, solutes drag along considerable amounts of water as they diffuse across channels. This results in low overall values of σ_s. The σ_s of HDO was extremely low. Its response to temperature was opposite to that for the σ_s of the alcohols. This suggested a stronger effect of temperature on the hydraulic (osmotic) than on the diffusive water flow across individual water channels, i.e. a differential sensitivity of different mechanisms to temperature. Received: 10 October 1996 / Accepted: 2 December 1996     

Studies on water transport through the sweet cherry fruit surface: characterizing conductance of the cuticular membrane using pericarp segments

Water conductance of the cuticular membrane (CM) of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated by monitoring water loss from segments of the outer pericarp excised from the cheek of the fruit. Segments consisted of epidermis, hypodermis and several cell layers of the mesocarp. Segments were mounted in stainless-steel diffusion cells with the mesocarp surface in contact with water, while the outer cuticular surface was exposed to dry silica (22 ± 1 °C). Conductance was calculated by dividing the amount of water transpired per unit area and time by the difference in water vapour concentration across the segment. Conductance values had a log normal distribution with a median of 1.15 × 10⁻⁴ m s⁻¹ (n=357). Transpiration increased linearly with time. Conductance remained constant and was not affected by metabolic inhibitors (1 mM NaN₃ or 0.1 mM carbonylcyanide m-chlorophenylhydrazone) or thickness of segments (range 0.8–2.8 mm). Storing fruit (up to 42 d, 1 °C) used as a source of segments had no consistent effect on conductance. Conductance of the CM increased from cheek (1.16 ± 0.10 × 10⁻⁴ m s⁻¹) to ventral suture (1.32 ± 0.07 × 10⁻⁴ m s⁻¹) and to stylar end (2.53 ± 0.17 × 10⁻⁴ m s⁻¹). There was a positive relationship (r²=0.066**; n=108) between conductance and stomatal density. From this relationship the cuticular conductance of a hypothetical astomatous CM was estimated to be 0.97 ± 0.09 × 10⁻⁴ m s⁻¹. Removal of epicuticular wax by stripping with cellulose acetate or extracting epicuticular plus cuticular wax by dipping in CHCl₃/methanol increased conductance 3.6- and 48.6-fold, respectively. Water fluxes increased with increasing temperature (range 10–39 °C) and energies of activation, calculated for the temperature range from 10 to 30 °C, were 64.8 ± 5.8 and 22.2 ± 5.0 kJ mol⁻¹ for flux and vapour-concentration-based conductance, respectively. Received: 23 March 2000 / Accepted: 28 July 2000     

Dose rate effect on micronuclei induction in human blood lymphocytes exposed to single pulse and multiple pulses of electrons

The effects of single pulses and multiple pulses of 7 MV electrons on micronuclei (MN) induction in cytokinesis-blocked human peripheral blood lymphocytes (PBLs) were investigated over a wide range of dose rates per pulse (instantaneous dose rate). PBLs were exposed to graded doses of 2, 3, 4, 6, and 8 Gy of single electron pulses of varying pulse widths at different dose rates per pulse, ranging from 1 × 10⁶ Gy s⁻¹ to 3.2 × 10⁸ Gy s⁻¹. Different dose rates per pulse were achieved by changing the dose per electron pulse by adjusting the beam current and pulse width. MN yields per unit absorbed dose after irradiation with single electron pulses were compared with those of multiple pulses of electrons. A significant decrease in the MN yield with increasing dose rates per pulse was observed, when dose was delivered by a single electron pulse. However, no reduction in the MN yield was observed when dose was delivered by multiple pulses of electrons. The decrease in the yield at high dose rates per pulse suggests possible radical recombination, which leads to decreased biological damage. Cellular response to the presence of very large numbers of chromosomal breaks may also alter the damage.     

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

Summary High yields of human hepatocytes (up to 23×10⁶ viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10⁻⁸ M insulin, and 10⁻⁸ M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg⁻¹·min⁻¹) similar to that reported for human liver. Insulin at 10⁻⁸ M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10⁻⁹ M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α₁-antitrypsin, α₁-antichymotrypsin, α₁-acid glycoprotein, haptoglobin, α₂-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10⁻⁹ M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg⁻¹ cell protein·min⁻¹, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg⁻¹). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.     

Kinetics of N-substituted phenothiazines and N-substituted phenoxazines oxidation catalyzed by fungal laccases

Laccase-catalyzed oxidation of N-substituted phenothiazines and N-substituted phenoxazines was investigated at pH 5.5 and 25°C. The recombinant laccase from Polyporus pinsitus (rPpL) and the laccase from Myceliophthora thermophila (rMtL) were used. The dependence of initial reaction rate on substrate concentration was analyzed by applying the laccase action scheme in which the laccase native intermediate (NI) reacts with a substrate forming reduced enzyme. The reduced laccase produces peroxide intermediate (PI) which in turn decays to the NI. The calculated constant (k_ox) values of the PI formation are (6.1±3.1)×10⁵ M⁻¹s⁻¹ for rPpL and (2.5±0.9)×10⁴ M⁻¹s⁻¹ for rMtL. The bimolecular constants of the reaction of the native intermediate with electron donor (kred) vary in the interval from 2.2×10⁵ to 2.1×10⁷ M⁻¹s⁻¹ for rPpL and from 1.3×10² to 1.8×10⁵ M^-1s⁻¹ for rMtL. The larger reactivity of rPpL in comparison to rMtL is associated with the higher redox potential of type I Cu of rPpL. The variation of k_red values for both laccases correlates with the change of the redox potential of substrates. Following outer sphere (Marcus) electron transfer mechanism the calculated activationless electron transfer rate and the apparent reorganization energy are 5.0×107 M⁻¹s⁻¹ and 0.29 eV, respectively.     

Differences among plant species in cuticular permeabilities and solute mobilities are not caused by differential size selectivities

Solute mobilities in cuticular membranes of six species (Hedera helix, Malus domestica, Populus alba, Pyrus communis, Stephanotis floribunda, Strophantus gratus) were measured using plant hormones, growth regulators and other organic model compounds varying in molar volumes from 99 to 349 mL · mol⁻¹ The dependence of mobilities (k*) on molar volume (V _x ) was exponential and could be described with equations of the type log k*=log k*⁰−^′ V _x . The y-intercepts (log k*⁰) represent mobilities of a hypothetical solute of zero molar volume. The parameter β′ is a measure of size selectivity of cuticular membranes and no differences among the six species were observed. At 25 °C the average β′ was 0.0095 mol · mL⁻¹. Solute mobility decreased by about a factor of 8.9 when molar volume increased by 100 mL · mol⁻¹ and the mobility of a compound with V _x  = 100 mL · mol⁻¹ was about 700-fold higher than the mobility of a compound with V _x  = 400 mL · mol⁻¹. Size selectivity decreased with increasing temperatures and for Strophantusβ′-values of 1.6 × 10⁻² to 8.0 × 10^-4 mol · mL⁻¹ were obtained for 10 and 30 °C, respectively. The-intercepts (log k*⁰) differed among plant species by 3 orders of magnitude and since size selectivity was the same for all species, solute mobilities for solutes having zero molar volumes were the sole cause for differences among species in solute mobilities and permeabilities. We argue that these differences in k*⁰ are related to tortuosity of the diffusion path. These results were used to derive an equation which predicts rates of cuticular penetration on the basis of k*⁰, the average size selectivity of 9.5 × 10⁻³ mol · mL⁻¹ and the driving forces of penetration. Received: 25 November 1997 / Accepted: 9 March 1998     

Carbon-nanotube-modified electrodes for the direct bioelectrochemistry of pseudoazurin

The bioelectrochemistry of the blue copper protein, pseudoazurin, at glassy carbon and platinum electrodes that were modified with single-wall carbon nanotubes (SWNTs) was investigated by multiple scan rate cyclic voltammetry. The protein showed reversible electrochemical behavior at both bare glassy carbon electrodes (GCEs) and SWNT-modified GCEs (SWNT|GCEs); however, direct electrochemistry was not observed at any of the platinum electrodes. The effect of the carbon nanotubes at the GCE was to amplify the current response 1000-fold (nA at bare GCE to μA at SWNT|GCE), increase the apparent diffusion coefficient D _app of the solution-borne protein by three orders of magnitude, from 1.35 × 10⁻¹¹ at bare GCE to 7.06 × 10⁻⁸ cm² s-1 at SWNT|GCE, and increase the heterogeneous electron transfer rate constant k _s threefold, from 1.7 × 10⁻² cm s⁻¹ at bare GCE to 5.3 × 10⁻² cm s⁻¹ at SWNT|GCE. Pseudoazurin was also found to spontaneously adsorb onto the nanotube-modified GCE surface. Well-resolved voltammograms indicating quasi-reversible faradaic responses were obtained for the adsorbed protein in phosphate buffer, with I _pc and I _pa values now greater than corresponding values for solution-borne pseudoazurin at SWNT|GCEs and with significantly reduced ΔE _p values. The largest electron transfer rate constant of 1.7 × 10⁻¹ cm s⁻¹ was achieved with adsorbed pseudoazurin at the SWNT|GCE surface in deaerated buffer solution consistent with its presumed role in anaerobic respiration of some bacteria.