Approximation of the Basic Reproduction Number <Emphasis Type="Italic">R</Emphasis><Subscript>0</Subscript> for Vector-Borne Diseases with a Periodic Vector Population

The main purpose of this paper is to give an approximate formula involving two terms for the basic reproduction number R ₀ of a vector-borne disease when the vector population has small seasonal fluctuations of the form p(t) = p ₀ (1+ε cos (ωt − φ)) with ε ≪ 1. The first term is similar to the case of a constant vector population p but with p replaced by the average vector population p ₀. The maximum correction due to the second term is (ε²/8)% and always tends to decrease R ₀. The basic reproduction number R ₀ is defined through the spectral radius of a linear integral operator. Four numerical methods for the computation of R ₀ are compared using as example a model for the 2005/2006 chikungunya epidemic in La Réunion. The approximate formula and the numerical methods can be used for many other epidemic models with seasonality. MSC 92D30 ⋅ 45C05 ⋅ 47A55     

A model for the effects of primary substrates on the kinetics of reductive dehalogenation

A kinetic model that describes substrate interactions during reductive dehalogenation reactions is developed. This model describes how the concentrations of primary electron-donor and -acceptor substrates affect the rates of reductive dehalogenation reactions. A basic model, which considers only exogenous electron-donor and -acceptor substrates, illustrates the fundamental interactions that affect reductive dehalogenation reaction kinetics. Because this basic model cannot accurately describe important phenomena, such as reductive dehalogenation that occurs in the absence of exogenous electron donors, it is expanded to include an endogenous electron donor and additional electron acceptor reactions. This general model more accurately reflects the behavior that has been observed for reductive dehalogenation reactions. Under most conditions, primary electron-donor substrates stimulate the reductive dehalogenation rate, while primary electron acceptors reduce the reaction rate. The effects of primary substrates are incorporated into the kinetic parameters for a Monod-like rate expression. The apparent maximum rate of reductive dehalogenation (q _{m, ap} ) and the apparent half-saturation concentration (K _ap ) increase as the electron donor concentration increases. The electron-acceptor concentration does not affect q _{m, ap} , but K _ap is directly proportional to its concentration.Definitions for model parameters RX halogenated aliphatic substrate - E-M ⁿ reduced dehalogenase - E-M ⁿ⁺² oxidized dehalogenase - [E-M ⁿ ] steady-state concentration of the reduced dehalogenase (moles of reduced dehalogenase per unit volume) - [E-M ⁿ⁺²] steady-state concentration of the oxidized dehalogenase (moles of reduced dehalogenase per unit volume) - DH₂ primary exogenous electron-donor substrate - A primary exogenous electron-acceptor substrate - A2 second primary exogenous electron-acceptor substrate - X biomass concentration (biomass per unit volume) - f fraction of biomass that is comprised of the dehalogenase (moles of dehalogenase per unit biomass) - stoichiometric coefficient for the reductive dehalogenation reaction (moles of dehalogenase oxidized per mole of halogenated substrate reduced) - stoichiometric coefficient for oxidation of the primary electron donor (moles of dehalogenase reduced per mole of donor oxidized) - stoichiometric coefficient for oxidation of the endogenous electron donor (moles of dehalogenase reduced per unit biomass oxidized) - stoichiometric coefficient for reduction of the primary electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - stoichiometric coefficient for reduction of the second electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - r _RX rate of the reductive dehalogenation reaction (moles of halogenated substrate reduced per unit volume per unit time) - r _d1 rate of oxidation of the primary exogenous electron donor (moles of donor oxidized per unit volume per unit time) - r _d2 rate of oxidation of the endogenous electron donor (biomass oxidized per unit volume per unit time) - r _a1 rate of reduction of the primary exogenous electron acceptor (moles of acceptor reduced per unit volume per unit time) - r _a2 rate of reduction of the second primary electron acceptor (moles of acceptor reduced per unit volume per unit time) - k _RX mixed second-order rate coefficient for the reductive dehalogenation reaction (volume per mole dehalogenase per unit time) - k _d1 mixed-second-order rate coefficient for oxidation of the primary electron donor (volume per mole dehalogenase per unit time) - k _d2 mixed-second-order rate coefficient for oxidation of the endogenous electron donor (volume per mole dehalogenase per unit time) - b first-order biomass decay coefficient (biomass oxidized per unit biomass per unit time) - k _a1 mixed-second-order rate coefficient for reduction of the primary electron acceptor (volume per mole dehalogenase per unit time) - k _a2 mixed-second-order rate coefficient for reduction of the second primary electron acceptor (volume per mole dehalogenase per unit time) - q _m,ap apparent maximum specific rate of reductive dehalogenation (moles of RX per unit biomass per unit time) - K _ap apparent half-saturation concentration for the halogenated aliphatic substrate (moles of RX per unit volume) - k _ap apparent pseudo-first-order rate coefficient for reductive dehalogenation (volume per unit biomass per unit time)     

Effect of hydromechanical forces on the production of filamentous haemagglutinin and pertussis toxin ofBordetella pertussis

Summary The production ofBordetella pertussis extracytoplasmic filamentous haemagglutinin (FHA) and pertussis toxin (PT) in a bioreactor under stirring conditions was studied in order to investigate the effect of hydromechanical forces on yields of both antigens. It was shown that FHA loses its haemagglutinin activity when the power transmitted by the agitator and the aerator per unit volume increases, whereas PT production is not affected. The loss of FHA activity can be explained by the action of shear forces on the filamentous structure of this antigen.Nomenclature C^* dissolved oxygen saturation concentration - C₁ dissolved oxygen concentration - D impeller diameter - power transmitted by the agitator and the aerator per unit of liquid volume - E_m maximum local energy dissipation rate per unit of liquid volume - K_La volumetric oxygen transfer coefficient - N impeller speed - Pg power input in aerated system - qO₂m maximum specific oxygen consumption rate - R_e Reynold number (D²N /) - VVM volume of air per volume of fermentation broth per minute - Xm maximum of biomass concentration - o Kolmogorov-microscale - fermentation broth viscosity - fermentation broth kinematic viscosity - fermentation broth density - expt experiment     

Partitioning of late gestation energy expenditure in ewes using indirect calorimetry and a linear regression approach

Abstract

Late gestation energy expenditure (EE_gest) originates from energy expenditure (EE) of development of conceptus (EE_conceptus) and EE of homeorhetic adaptation of metabolism (EE_homeorhetic). Even though EE_gest is relatively easy to quantify, its partitioning is problematic. In the present study metabolizable energy (ME) intake ranges for twin-bearing ewes were 220 – 440, 350 – 700, 350 – 900 kJ per metabolic body weight (W^0.75) at week seven, five, two pre-partum respectively. Indirect calorimetry and a linear regression approach were used to quantify EE_gest and then partition to EE_conceptus and EE_homeorhetic. Energy expenditure of basal metabolism of the non-gravid tissues (EE_bmng), derived from the intercept of the linear regression equation of retained energy [kJ/W^0.75] and ME intake [kJ/W^0.75], was 298 [kJ/W^0.75]. Values of the intercepts of the regression equations at week seven, five, and two pre-partum were 311, 398, and 451 [kJ/W^0.75], respectively. The difference between the intercepts for different weeks was used to calculate EE_homeorhetic. The remaining part of EE_gest was considered to be EE_conceptus. In conclusion, the good agreement between our values of EE_conceptus and those in the literature indicates the method's validity.     

Leaf water relations characteristics of differently potassium fertilized and watered field grown barley plants

Seasonal leaf water relations characteristics were studied in fully irrigated spring barley (Hordeum distichum L. cv. Gunnar) fertilized at low (50 kg K ha⁻¹) or high (200 kg K ha⁻¹) levels of potassium applied as KCl. The investigation was undertaken from about 14 days before anthesis until the milk ripe stage in leaves of different position and age. Additionally, the effects of severe water stress on leaf water relations were studied in the middle of the grain filling period in spring barley (cv. Alis). The leaf water relations characteristics were determined by the pressure volume (PV) technique. Water relations of fully irrigated plants were compared in leaf No 7 with the water relations of slowly droughted plants (cv. Alis). Leaf osmotic potential at full turgor (ψ _π ¹⁰⁰ ) decreased 0.1 to 0.3 MPa in droughted leaves indicating a limited osmotic adjustment due to solute accumulation. The leaf osmotic potential at zero turgor (ψ _π ⁰ ) was about −2.2 MPa in fully irrigated plants and −2.6 MPa in droughted plants. The relative water content at zero turgor (R⁰) decreased 0.1 unit in severely droughted leaves. The ratio of turgid leaf weight to dry weight (TW/DW) tended to be increased by drought. The tissue modulus of elasticity (ε) decreased in droughted plants and together with osmotic adjustment mediated turgor maintenance during drought. A similar response to drought was found in low and high K plants except that the R⁰ and ε values tended to be higher in the high K plants. Conclusively, during drought limited osmotic adjustment and increase in elasticity of the leaf tissue mediated turgor maintenance. These effects were only slightly modified by high potassium application. The seasonal analysis in fully irrigated plants (cv. Gunnar) showed that within about 14 days from leaf emergence ψ _π ¹⁰⁰ decreased from about −0.9 to −1.6 MPa in leaf No 7 (counting the first leaf to emerge as number one) and from about −1.1 to −1.9 MPa in leaf No 8 (the flag leaf) due to solute accumulation. A similar decrease took place in ψ _π ⁰ except that the level of ψ _π ⁰ was displaced to a lower level of about 0.2 to 0.3 MPa. Both ψ _π ¹⁰⁰ and ψ _π ⁰ tended to be 0.05 to 0.10 MPa lower in high K than in low K plants. R⁰ was about 0.8 to 0.9 and was independent of leaf position and age, but tended to be highest in high K plants. The TW/DW ratio decreased from about 5.5 in leaf No 6 to 4.5 in leaf No 7 and 3.8 in leaf No 8. The TW/DW ratio was 4 to 10% higher in high K than in low K plants indicating larger leaf cell size in the former. The apoplastic water content (V_a) at full turgor constituted about 15% in leaf No 7. ε was maximum at full turgor and varied from about 11 to 34 MPa. ε tended to be higher in high K plants. Conclusively, in fully watered plants an ontogenetically determined accumulation of solutes (probably organic as discussed) occurred in the leaves independent of K application. The main effect of high K application on water relations was an increase in leaf water content and a slight decrease in leaf ψ_π. The effect of K status on growth and drought resistance is discussed.     

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50–100 mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml^-1 Cellulase R-10 and Macerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25×10⁶ viable protoplasts per g fresh weight and their size ranged from 12 to 44 m. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10×10⁵ protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [¹⁴C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN₃, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake.     

Effects of nutrient enrichment on the release of dissolved organic carbon and nitrogen by the scleractinian coral Montipora digitata

The effects of nutrient enrichment on the release of dissolved organic carbon and nitrogen (DOC and DON, respectively) from the coral Montipora digitata were investigated in the laboratory. Nitrate (NO₃ ⁻) and phosphate (PO₄ ³⁻) were supplied to the aquarium to get the final concentrations of 10 and 0.5 μmol l⁻¹, respectively, and the corals were incubated for 8 days. The release rate of DON per unit coral surface area significantly decreased after the nutrient enrichment, while the release rate of DOC was constant. Because the chlorophyll a (chl a) content of zooxanthellae per unit surface area increased, the release rate of DOC significantly decreased when normalized to unit chl a. These results suggested that the incorporation of NO₃ ⁻ and PO₄ ³⁻ stimulated the synthesis of new cellular components in the coral colonies and consequently, reduced extracellular release of DOC and DON. Actually, significant increase in N and P contents relative to C content was observed in the coral’s tissue after the nutrient enrichment. The present study has concluded that inorganic nutrient enrichment not only affects coral-algal metabolism inside the colony but also affects a microbial community around the coral because the organic matter released from corals functions as energy carrier in the coral reef ecosystem.     

Production of bialaphos-resistant Nierembergia repens by electroporation

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.     

Biochemical and physiological properties of a protein inducing protoplast release during conjugation in theClosterium peracerosum-strigosum-littorale complex

When mating-type minus (mt⁻) and plus (mt⁺) cells of theClosterium peracerosum-strigosum-littorale complex were mixed together in a nitrogen-deficient mating medium, cells of both types released protoplasts, this release being the first step in the process of conjugation. Release of protoplasts by mt⁻ cells also proceeded without pairing in a medium in which mt⁻ and mt⁺ cells had previously been cultured together. A protein with the ability to induce the release of protoplasts was purified from this medium by sequential column-chromatographic steps, and named PR-IP (protoplast-release-inducing protein). The PR-IP had an apparent molecular mass (M_r) of 95000 on gel filtration and could be separated into several isoforms by anion-exchange chromatography. Each isoform consisted of two glycopolypeptides of M_rs 42000 and 19000, while the deglycosylated polypeptides had M_rs of 34000 and 18000, respectively. From an analysis of dose-response curves, the numbers of PR-IP molecules required for the release of a protoplast by a single cell was calculated as 1.5·10⁹ and the concentration required for 50% of the maximum response (ED₅₀) as 4.1·10⁻⁹M. We suggest that the PR-IP is a biologically active glycoprotein which induces the release of gametic protoplasts from mt⁻ cells of thisClosterium complex.     

Local Osmosis and Isotonic Transport

Osmotically driven water flow, u (cm/s), between two solutions of identical osmolarity, c_o (300 mM in mammals), has a theoretical isotonic maximum given by u = j/c_o, where j (moles/cm²/s) is the rate of salt transport. In many experimental studies, transport was found to be indistinguishable from isotonic. The purpose of this work is to investigate the conditions for u to approach isotonic. A necessary condition is that the membrane salt/water permeability ratio, ε, must be small: typical physiological values are ε = 10⁻³ to 10⁻⁵, so ε is generally small but this is not sufficient to guarantee near-isotonic transport. If we consider the simplest model of two series membranes, which secrete a tear or drop of sweat (i.e., there are no externally-imposed boundary conditions on the secretion), diffusion is negligible and the predicted osmolarities are: basal = c_o, intracellular ≈ (1 + ε)c_o, secretion ≈ (1 + 2ε)c_o, and u ≈ (1 − 2ε)j/c_o. Note that this model is also appropriate when the transported solution is experimentally collected. Thus, in the absence of external boundary conditions, transport is experimentally indistinguishable from isotonic. However, if external boundary conditions set salt concentrations to c_o on both sides of the epithelium, then fluid transport depends on distributed osmotic gradients in lateral spaces. If lateral spaces are too short and wide, diffusion dominates convection, reduces osmotic gradients and fluid flow is significantly less than isotonic. Moreover, because apical and basolateral membrane water fluxes are linked by the intracellular osmolarity, water flow is maximum when the total water permeability of basolateral membranes equals that of apical membranes. In the context of the renal proximal tubule, data suggest it is transporting at near optimal conditions. Nevertheless, typical physiological values suggest the newly filtered fluid is reabsorbed at a rate u ≈ 0.86 j/c_o, so a hypertonic solution is being reabsorbed. The osmolarity of the filtrate c_F (M) will therefore diminish with distance from the site of filtration (the glomerulus) until the solution being transported is isotonic with the filtrate, u = j/c_F.With this steady-state condition, the distributed model becomes approximately equivalent to two membranes in series. The osmolarities are now: c_F ≈ (1 − 2ε)j/c_o, intracellular ≈ (1 − ε)c_o, lateral spaces ≈ c_o, and u ≈(1 + 2ε)j/c_o. The change in c_F is predicted to occur with a length constant of about 0.3 cm. Thus, membrane transport tends to adjust transmembrane osmotic gradients toward εc_o, which induces water flow that is isotonic to within order ε. These findings provide a plausible hypothesis on how the proximal tubule or other epithelia appear to transport an isotonic solution.     

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation

3 S₄]⁺, S=1/2, composed of three, antiferromagnetically coupled high-spin ferric ions) by continuous wave (CW) and pulsed EPR techniques: Azotobacter vinelandii ferredoxin I, Desulfovibrio gigas ferredoxin II, and the 3Fe forms of Pyrococcus furiosus ferredoxin and aconitase. The 35 GHz (Q-band) CW EPR signals are simulated to yield experimental g tensors, which either had not been reported, or had been reported only at X-band microwave frequency. Pulsed X- and Q-band EPR techniques are used to determine electron spin-lattice (T ₁, longitudinal) relaxation times at several positions on the samples' EPR envelope over the temperature range 2–4.2 K. The T ₁ values vary sharply across the EPR envelope, a reflection of the fact that the envelope results from a distribution in cluster properties, as seen earlier as a distribution in g ₃ values and in ⁵⁷ Fe hyperfine interactions, as detected by electron nuclear double resonance spectroscopy. The temperature dependence of 1/T ₁ is analyzed in terms of the Orbach mechanism, with relaxation dominated by resonant two-phonon transitions to a doublet excited state at ∼20 cm⁻¹ above the doublet ground state for all four of these 3Fe proteins. The experimental EPR data are combined with previously reported ⁵⁷Fe hyperfine data to determine electronic spin exchange-coupling within the clusters, following the model of Kent et al. Their model defines the coupling parameters as follows: J ₁₃=J, J ₁₂=J(1+ε′), J ₂₃=J(1+ε), where J _ij is the isotropic exchange coupling between ferric ions i and j, and ε and ε′ are measures of coupling inequivalence. We have extended their theory to include the effects of ε′≠0 and thus derived an exact expression for the energy of the doublet excited state for any ε, ε′. This excited state energy corresponds roughly to ε J and is in the range 5–10 cm⁻¹ for each of these four 3Fe proteins. This magnitude of the product ε J, determined by our time-domain relaxation studies in the temperature range 2–4 K, is the same as that obtained from three other distinct types of study: CW EPR studies of spin relaxation in the range 5.5–50 K, NMR studies in the range 293–303 K, and static susceptibility measurements in the range 1.8–200 K. We suggest that an apparent disagreement as to the individual values of J and ε be resolved in favor of the values obtained by susceptibility and NMR (J≳200 cm⁻¹ and ε≳0.02 cm⁻¹ ), as opposed to a smaller J and larger ε as suggested in CW EPR studies. However, we note that this resolution casts doubt on the accepted theoretical model for describing the distribution in magnetic properties of 3Fe clusters. Received: 23 December 1999 / Accepted: 8 March 2000     

Light-Enhanced Uptake of Metabolites in Mesophyll Protoplasts: Evidence for a Novel Pyruvate Transport System

3 ) and sorghum (C₄) leaves for the measurements of osmotic volume change and metabolite uptake. We first investigated whether the silicone oil layer filtering centrifugation method could be applied to the protoplasts. The density of the silicone oil was optimized (ρ =1.026) and 0.5M betaine was chosen as an osmoticum in the protoplast suspending medium. By using [¹⁴C] sorbitol and [¹⁴C] inulin as the marker of the medium carried over into the pellet, protoplast osmotic or internal volume was estimated to be 200–300 μl (mg Chl)⁻¹, with the medium space in the pellet of 8–15 μl (mg Chl)⁻¹. Lowering of the osmotic pressure of the medium induced protoplast swelling as expected. Light also induced swelling. Using this system, we could detect light-enhanced uptake of ascorbate, glutamate and pyruvate in both barley and sorghum protoplasts. Pyruvate uptake was far higher in barley than in sorghum and inhibited by various inhibitors, showed saturation kinetics and, therefore, seemed to be mediated by a translocator protein. Received 10 August 1999/ Accepted in revised form 6 December 1999     

Isolation of NADH Oxidation System from the Plasmalemma of Corn Root Protoplasts

A plasmalemma-bound NADH oxidation system (Lin 1982 Proc Natl Acad Sci USA 79: 3773-3776) in corn root protoplasts was isolated by a mild treatment of intact protoplasts with trypsin. The majority of NADH stimulated O₂ consumption activity of the protoplasts could be recovered in the supernatant isolated from the intact protoplasts which have been treated with trypsin. The activation energy of NADH oxidation in the supernatant is similar to that of the intact protoplasts (8.7 versus 9.4 kilocalories per mole per degree). Unlike that of the intact protoplasts, an Arrhenius plot of the temperature response (from 5 to 25°C) of the activity in the supernatant shows no transition suggestive of a dissociation of the enzyme from the membrane. Trypsin treatment did not affect K⁺ uptake into cell volume of the protoplast. However, the NADH-stimulated K⁺ uptake and the increase of cell volume were greatly reduced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitated protein from the supernatant showed one extra peptide band with ~42 kilodalton molecular weight.     

Herbicide-resistant transgenic creeping bentgrass plants obtained by electroporation using an altered buffer

Modification of an electroporation buffer using Ca(NO₃)₂ and elevated pH (9–10) appeared to have a favorable effect on gene transfer to creeping bentgrass (Agrostis palustris Huds.) Penncross protoplasts, resulting in an increase in the transformation frequency of about twofold. Following electroporation with the plasmid pARK22 containing the bar gene, a total of 278 bialaphos-resistant cell colonies were obtained from four experiments. The bialaphos-resistant regenerants proved to be transgenic by Southern hybridization of the amplified DNA. All the tested transgenic plants showed herbicide (HERBIE) resistance at the field rate of 0.5–1% (vol/vol). Ammonia contents in leaves after spraying with the herbicide increased less in transgenic plants than in untransformed control plants. Received: 10 February 1998 / Revision received: 8 April 1998 / Accepted: 24 April 1998     

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     

Transformation of sweet potato tissues with green-fluorescent protein gene

Summary The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×10⁶ protoplasts ml⁻¹ was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm⁻¹. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.     

[Pt(dien)]2+ migrates intramolecularly from methionine S to imidazole Nz 2 in the peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh

The pH- and time-dependent reaction of [Pt(dien)(H₂O)]²⁺ with the methionine- and histidine-containing peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh at 313 K has been investigated by HPLC and NMR spectroscopy. For both peptides, initial relatively rapid formation of the kinetically favoured methionine S-bound complex is followed by slow intramolecular migration of the [Pt(dien)]²⁺ fragment to imidazole N_{ε 2} (or, in the case of H-His-Gly-Met-OH, to a much lesser extent to the competing imidazole N_{δ 1}) of the histidine side chain over a period of 500 h. Time-dependent studies for the pentapeptide at pH 8.0 demonstrate that this isomerization can take place by either direct S→N_{ε 2} migration or by a two-step mechanism involving initial N_{ε 2} coordination of a second [Pt(dien)]²⁺ fragment and subsequent cleavage of the orginal Pt-S bond in the resulting dinuclear complex. The rate of κS/κN _{ε 2} isomerization is markedly reduced on lowering the pH to 5.1. Received: 26 February 1999 / Accepted: 14 April 1999     

Up-regulation of cyclic electron flow and down-regulation of linear electron flow in antisense-<Emphasis Type="Italic">rca</Emphasis> mutant rice

To investigate how excess excitation energy is dissipated in a ribulose-1,5-bisphospate carboxylase/oxygenase activase antisense transgenic rice with net photosynthetic rate (P _N) half of that of wild type parent, we measured the response curve of P _N to intercellular CO₂ concentration (C _i), electron transport rate (ETR), quantum yield of open photosystem 2 (PS2) reaction centres under irradiation (F_v′/F_m′), efficiency of total PS2 centres (Φ_PS2), photochemical (q_P) and non-photochemical quenching (NPQ), post-irradiation transient increase in chlorophyll (Chl) fluorescence (PITICF), and P700⁺ re-reduction. Carboxylation efficiency dependence on C _i, ETR at saturation irradiance, and F_v′/F_m′, Φ_PS2, and q_P under the irradiation were significantly lower in the mutant. However, NPQ, energy-dependent quenching (q_E), PITICF, and P700⁺ re-reduction were significantly higher in the mutant. Hence the mutant down-regulates linear ETR and stimulates cyclic electron flow around PS1, which may generate the ΔpH to support NPQ and q_E for dissipation of excess excitation energy.     

Electroporative deformation of salt filled lipid vesicles

Membrane electroporation, vesicle shape deformation and aggregation of small, NaCl-filled lipid vesicles (of radius a = 50 nm) in DC electric fields was characterized using conductometric and turbidimetrical data. At pulse durations t_E≤ 55 ± 5 ms the increase in the conductivity of the vesicle suspension is due to the field-induced efflux of electrolyte through membrane electropores. Membrane electroporation and Maxwell stress on the vesicle membrane lead to vesicle elongation concomitant with small volume reduction (up to 0.6% in an electric field of E = 1 MV m^–1). At t_E > 55 ± 5 ms, further increases in the conductivity and the optical density suggest electroaggregation and electrofusion of vesicles. The conductivity changes after the electric pulse termination reflect salt ion efflux through slowly resealing electropores. The analysis of the volume reduction kinetics yields the bending rigidity κ = (4.1 ± 0.3) ⋅ 10^–20 J of the vesicle membrane. If the flow of Na⁺ and Cl^– ions from the vesicle interior is treated in terms of Hagen-Poiseuille's equation, the number of permeable electropores is N = 39 per vesicle with mean pore radius r_p = 0.85 ± 0.05 nm at E = 1 MVm^–1 and t_E≤ 55 ± 5 ms. The turbidimetric and conductometric data suggest that small lipid vesicles (a ≤ 50 nm) are not associated with extensive membrane thermal undulations or superstructures. In particular with respect to membrane curvature, the vesicle results are suggestive for the design and optimization of electroporative delivery of drugs and genes to cell tissue at small field strengths (≤1 MVm^–1) and large pulse durations (≤100 ms). Received: 8 July 1997 / Accepted: 15 September 1997     

Characterization of transgenic rice plants that express rgp1, the gene for a small GTP-binding protein from rice

 The rgp1 gene, which encodes a small GTP-binding protein from rice, was introduced into rice protoplasts by electroporation. Transformed protoplasts were cultured on liquid protoplast-culture medium for 1 month, and then cells that had proliferated were transferred to a selection medium that contained 50 mg/l hygromycin B. Among 50 colonies that were selected and transferred to regeneration medium, 3 colonies generated shoots. However, two of the three shoots failed to form roots and ceased growing. A single regenerated shoot that formed roots was planted in soil and transferred to a greenhouse. Southern hybridization showed that the regenerated plant harbored a single copy of the introduced gene. The transformant (T₀) plant was shorter than the controls, it developed three times as many tillers as controls, it developed three times as many tillers as control plants but it produced mostly sterile seeds. In a test of hygromycin resistances, viable seeds segregated into resistant and sensitive seedings at a ratio of approximately 1 : 3. The progeny (T₁) plants were short with many tillers, and some produced seeds normally. The T₂ seedlings grew more rapidly than control seedlings for the first 28 days after germination, but control plants subsequently outgrew the T₂ plants. Northern blotting analysis revealed that the rgp1 gene in T₂ plants was expressed consitutively throughout all developmental stages. The results suggest that the observed phenotypic changes were due to expression of the exogenous rgp1 gene. Received: 21 September 1997/Accepted: 31 March 1998