Removal of Linear and Branched Alkylphenols from Aqueous Solutions with Horseradish Peroxidase

Alkylphenols were effectively treated with horseradish peroxidase at pH 7.0 and 30 °C in the presence of H₂O₂ and poly(ethylene glycol) irrespective of the relative position or isomeric form of the alkyl chains. Water-insoluble oligomer precipitates were readily filtered out after enzymatic treatment, and transparent and colorless solutions were obtained for all p- and m-alkylphenols used.     

Protein mass transfer studies on a spray column using the PEG-Reppal PES 100 aqueous two-phase system

The characterization of Bovine Serum Albumin mass transfer mechanisms in a spray column using an aqueous two-phase system composed of poly(ethylene glycol) and a modified starch-Reppal PES 100-is done. The poly(ethylene glycol) rich phase is used as the dispersed phase and protein transfer takes place from the dispersed phase to the continuous phase. The effect of dispersed phase superficial velocity, system composition, continuous phase height and distribution system design on either overall protein mass transfer coefficient or column hold-up is described. It is shown that continuous phase superficial velocity and phase composition are the main controlling factors for protein transfer. It is also observed that, with the tested system, only at very low dispersed phase superficial velocities is it possible to operate the spray column as an extraction column. In this system the upper operating limit of the dispersed phase velocity is ten times smaller than in other aqueous two-phase systems.List of Symbols ATPS Aqueous Two-Phase System - BSA Bovine Serum Albumin - C _i kg m^–3 inlet dispersed phase protein concentration - C ₀ kg m^–3 outlet dispersed phase protein concentration - C _d kg m^–3 dispersed phase protein concentration - C _c kg m^–3 continuous phase protein concentration - D m column internal diameter - H hold-up - h, h _d m dispersion height - h ₀ m initial dispersion height (initial continuous phase height) - k _da s^–1 overall mass transfer coefficient - m protein partition coefficient - n number of holes of distribution system - PEG Poly(ethylene glycol) - Q m³ s^–1 dispersed phase volumetric flow rate - S m² column internal area - V m³ dispersion volume A. Venâncio was supported by a JNICT (Junta Nacional de Investigaçäo Científica e Tecnológica) grant.     

Stabilization method of an alkaline protease from inactivation by heat,SDS and hydrogen peroxide

An investigation was carried out to evaluate the protective effect of polyhydric alcohols, such as propylene glycol and glycerol on the inactivation of an alkaline protease by sodium dodecyl sulfate (SDS) and H₂O₂. Addition of polyols increased the stability of a Bacillus clausii I-52 alkaline protease towards not only the thermal-induced, but also the SDS and H₂O₂-induced inactivation. Among the polyols examined, the best results were obtained with propylene glycol. The half-life of the enzyme was increased by 43- and >105-fold by the addition of 10% (v/v) propylene glycol to the enzyme preparations containing 5% (w/v) SDS and 5% (v/v) H₂O₂ at 50 °C, respectively. Besides the protection effect of propylene glycol from enzyme inactivation by SDS and H₂O₂, it also improved the hydrolytic efficiency towards substrate like BSA during the protease reaction containing SDS or H₂O₂. This result suggests that propylene glycol has a significant potential as a good stabilizer of an alkaline protease preparation, which finds use as an additive in industrial applications, especially, the detergent industry.     

Effects of deuterium on the thermal stability of the poly A-poly U helix

The effect of deuterium on the thermal stability of the polynecleotide double helix formed by the homopolymers polyadenylie acid (poly A)and polyuridylic acid (poly U)has been invertigated by measuring the best capacity as a function of temperature in an automatic scanning adiabatic calorimeter. Hydrogen-bounded and deuterium-bonded helical conformations of the polynecleotides used have been melted in H₂O and D₂O₂ respectively, as solvent. Within the limits of experimental error, there is no dfference in the measured enthallpy change accompanying the helix-random coil transition. The enthalpy change ΔH is 6.6 Kcal/MBP ub any case. The half-conversion temperatures T_m differ by two degrees. T_m for poly (AU) in H₂O is 45.8°C, T_m for poly (AU) in D₂is 47.7.°C.     

Smart hydrogels for in situ generated implants

The objective of this study was to explore the use of reverse thermo-responsive (RTG) polymers for generating implants at their site of performance, following minimally invasive surgical procedures. Aiming at combining syringability and enhanced mechanical properties, a new family of injectable RTG-displaying polymers that exhibit improved mechanical properties was created, following two different strategies: (1) to synthesize high-molecular-weight polymers by covalenty joining poly(ethylene glycol) and poly(propylene glycol) chains using phosgene as the coupling molecule and (2) to cross-link poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblocks after end-capping them with triethoxysilane or methacrylate reactive groups. While the methacrylates cross-linked rapidly, the triethoxysilane groups enabled the system to cross-link gradually over time. The chain-extended PEO/PPO copolymers had molecular weights in the 39 000-54 000 interval and exhibited improved mechanical properties. Reverse thermo-responsive systems displaying gradually increasing mechanical properties were generated by cross-linking triethoxysilane-capped (EO)(99)-(PO)(67)-(EO)(99) (F127) triblocks. Over time, the ethoxysilane groups hydrolyzed and created silanol moieties that subsequently condensated. With the aim of further improving their mechanical behavior, F127 triblocks were reacted with methacryloyl chloride and the resulting dimethacrylate was subsequently cross-linked in an aqueous solution at 37 degrees C. The effect of the concentration of the F127 dimethacrylate on the mechanical properties and the porous structure of the cross-linked matrixes produced was assessed. Rheometric studies revealed that the cross-linked hydrogels attained remarkable mechanical properties and allowed the engineering of robust macroscopic constructs, such as large tubular structures. The microporosity of the matrixes produced was studied by scanning electron microscopy. Monolayered conduits as well as structures comprising two and three layers were engineered in vitro, and their compliance and burst strength were determined.     

Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol)

Hybrid gels constructed from proteins and polymers have attracted a wide range of attention in the field of biomedicine and bioengineering. We report herein the enzymatic preparation of polymer–protein hybrid hydrogels composed of terminally bis-functionalized linear poly(ethylene glycol) (PEG) and streptavidin (SA). PEG was conjugated with tyramine to introduce terminal phenolic hydroxyl (Ph-OH) groups. A peptide tag containing a tyrosine residue (G₄Y-tag) was genetically introduced at the C-terminus of SA. The Ph-OH-modified PEG and G₄Y-tagged SA (SA-G₄Y) were treated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂) to yield (PEG-Ph-OH)–(SA-G₄Y) hybrid gels. Biotinylated enhanced green fluorescent protein (biotin-EGFP) was selectively captured in the obtained hybrid gels, indicating that SA-G₄Y retained its biological function. The amount of biotin-EGFP immobilized in the hybrid gels depended on the concentration of SA-G₄Y. In addition, biotinylated bacterial alkaline phosphatase (biotin-BAP) was immobilized in the hybrid gel. The immobilized biotin-BAP exhibited more than 95% of the initial activity after 5 rounds of recycling. The results suggest the facile functionalization of the hybrid gel with a variety of biotinylated functional molecules.     

Upsurge in respiration and peroxide formation in potato tubers as influenced by ethylene, propylene, and cyanide

A continuous application of ethylene (10 μl/l) and propylene (500 μl/l) to potato tubers (Solanum tuberosum L.) resulted in an upsurge of respiration and a concomitant rise in peroxides. When applied in 100% O₂, the effect of ethylene and propylene on respiration and peroxide formation was augmented. Hydrogen cyanide (500 μl/l) mimicked the action of ethylene and propylene inducing a respiratory rise and a corresponding increase in peroxides. As with ethylene, the effect of HCN was augmented in high O₂ tensions. The results support the suggestion that ethylene activates the cyanide-insensitive respiratory pathway.     

An Efficient Method for Separating Ascospores from Sporulating Cultures in Saccharomyces cerevisiae by Hydrostatic Pressure

A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent K_ms were 2.96 m and 28.6 mm, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent K_m for glycolaldehyde was found to be 83.3 mm. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 m ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.     

Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers.

Four new poly(hydroxyethylaspartamide)-based copolymers bearing (a) poly(ethylene glycol) 2000, (b) poly(ethylene glycol) 5000, (c) poly(ethylene glycol) 2000 and hexadecylalkyl, (d) poly(ethylene glycol) 5000 and hexadecylalkyle, as pendant groups were synthesised. The copolymers were obtained by partial aminolysis of polysuccinimide with poly(ethylene glycol) and hexadecylalkyl amino derivatives followed by reaction with ethanolamine. Naked polyhydroxyaspartamide was obtained by polysuccinimide reaction with ethanolamine. The nuclear magnetic resonance, infrared, light scattering and elemental analysis allowed for the extensive physico-chemical characterisation of the carriers. The molecular mass of all the polymers was in the range of 27000-34000 Da, and the polydispersivity was in the range of 1.5-1.7. By intravenous injection to mice bearing a solid tumour, all the polymeric carriers displayed a bi-compartmental pharmacokinetic behaviour. Both the poly(ethylene glycol) and the hexadecylalkyle conjugation prolonged and enhanced the distribution phase of poly(hydroxyethylaspartamide). The poly(ethylene glycol) conjugation was found to promote the carrier elimination by kidney ultrafiltration and to prevent partially the accumulation in the spleen and in the liver. The poly(ethylene glycol)/hexadecylalkyle conjugates localised preferentially in the liver were over 30% of the dose/g of tissue was determined after 144 h from administration. In the tumour all the polymers displayed a relevant accumulation that significantly increased throughout the time to reach high concentrations after 24 h. In particular, the poly(ethylene glycol)/hexadecylalkyle conjugates achieved a concentration of 15-25% of the dose/g of tissue after 24 h from administration that was maintained up to 144 h.     

Effect of poly(ethylene glycol) on phospholipid hydration and polarity of the external phase

The hydration properties of phosphatidylcholine (PC)/water dispersions on the addition of poly(ethylene glycol) were studied by means of ²H-NMR. The quadrupole splittings and their temperature dependences correspond to measurements of PC/water dispersions at low water content. It is concluded that the bound water is partly extracted by poly(ethylene glycol) but the binding properties of the water in the inner hydration shell of about five water molecules are not changed. The ability of some phospholipid/water dispersions to undergo phase transitions to nonlamellar structures upon dehydration is discussed. Dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine do not form nonlamellar structures on addition of purified poly(ethylene glycol), as was demonstrated by means of ³¹P-NMR. Poly(ethylene glycol) decreases the polarity of the aqueous phase and the partition of hydrophobic molecules between the membrane and the external phase is changed. This was demonstrated using the excimer fluorescence of pyrene in a ghost suspension. It is suggested that the changes in polarity and hydration on the addition of poly(ethylene glycol) can contribute to the alterations in the membrane surface observed under conditions of membrane contact and fusion.     

A Direct Synthesis of Unsaturated Uracil Nucleosides from Silylated Uracil and Triacetylglucal

A kinetic analysis of splitting oligomeric substrates by poly(β-D-mannuronate)lyases (alginate lyases I, SP1 and SP2) from a marine mollusk was done. Monomer and oligomers of mannuronate and guluronate were prepared by hydrolyzing poly β-1,4-D-mannuronate and poly α-1,4-L-guluronate from alginate with H₂SO₄, respectively, and thereafter by gel filtration on a Bio-Gel P-2 column. Alginate lyases I apparently did not act on the trimer of mannuronate but did on the tetramer or those longer than that, indicating the increased k_cad/K_m with increasing polymerization degree. The kinetic analyses suggest that the size of the subsite structure of the enzymes is most likely to be able to bind the linear pentamer of mannuronate units.     

Enantioselective oxidations catalyzed by diketocamphane monooxygenase from Pseudomonas putida with macromolecular NAD in a membrane reactor

Summary Several sulfides and bicyclo[3.2.0]hept-2-en-6-one were enantioselectively oxidized to the corresponding sulfoxides and oxa lactones by a crude preparation of the two diketocamphane monooxygenases from Pseudomonas putida. The reactions were carried out in a membrane reactor with the use of poly(ethylene glycol)-N⁶-(2-aminoethyl)-NAD and coenzyme regeneration by the formate/formate dehydrogenase system.     

Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells

Summary Thein vivo andin vitro effects of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on calcium uptake by isolated chick duodenal cells were studied.In vivo, 1,25-(OH)₂D₃ given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion.In vitro, pre-incubation of 1,25-(OH)₂D₃ with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10^–15 m, was maximal at 10^–13 m, and was diminished at higher (10^–11 m) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D₃ analogs was 1,25-(OH)₂D₃=1-(OH)D₃>25-(OH)D₃>1,24,25-(OH)₃D₃>24,25-(OH)₂D₃>D₃. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)₂D₃ was five orders of magnitude greater than D₃. Kinetically, 1,25-(OH)₂D₃ increased theV _max of calcium uptake; the affinity for calcium (K _m=0.54mm) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)₂D₃,in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect of calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)₂D₃. These findings suggest that thein vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)₂D₃ induction of calcium transport and specific biochemical correlates.     

Defining the optimal cryoprotectant and concentration for cryopreservation of limbal stem cells

Limbal stem cell (LSC) deficiency causes progressive loss of vision but may be treated by transplant of autologous LSCs. Cryopreservation has the potential to indefinitely extend the lifespan of LSCs allowing re-transplant in case of graft failure. In this study, we aimed to identify the optimal cryoprotectant and cryoprotectant concentration for LSC cultures. Suspension cultures derived from cadaveric corneoscleral rims were cooled to 4 °C with Me₂SO, propylene glycol or ethylene glycol at a concentration of 5%, 10% or 15%. Cell tolerance was measured in terms of membrane integrity, colony-forming efficiency and alamarBlue^® reduction. Increasing cryoprotectant concentration above 5% reduced membrane integrity, metabolism and colony-forming efficiency. Cryoprotectant choice did not significantly influence these characteristics. Cells demonstrating Side Population were maintained after cryopreservation with 5% propylene glycol in vapour phase liquid nitrogen for 1 week, indicating that cryopreservation of LSCs with relatively low cryoprotectant concentration (5%) has promise in low-temperature eye banking.     

Microphytobenthos activity and fluxes at the sediment-water interface: interactions and spatial variability

In this study oxygen and nutrient fluxes and denitrification rates across the sediment-water interface were measured via intact core incubations with a twofold aim: show whether microphytobenthos activity affects these processes and analyse the dispersion of replicate measurements. Eighteen intact sediment cores (i.d. 8 cm) were randomly sampled from a shallow microtidal brackish pond at Tjarno, on the west coast of Sweden, and were incubated in light and in darkness simulating in situ conditions. During incubation O₂, inorganic N and SiO₂ fluxes and denitrification rates (isotope pairing) were measured. Assuming mean values of 18 cores as best estimate of true average (BEA), the accuracy of O₂, NH₄ ⁺, NO₃ ^- and SiO₂ fluxes calculated for an increasing number of subsamples was tested. At the investigated site, microalgae strongly influenced benthic O₂, inorganic N and SiO₂ fluxes and coupled (D_n) and uncoupled (D_w) denitrification through their photosynthetic activity. In the shift between dark and light conditions NH₄ ⁺ and SiO₂ effluxes (60 and 110 µmol m^-2h^-1) and D_n (5 µmol m^-2 h^-1) dropped to zero, NO₃ ^- uptake (70 µmol m^-2 h^-1) showed a 30% increase, while D_w (20 µmol m^-2 h^-1) showed an 80% decrease. For O₂ and NO₃ ^- dark fluxes, 4 core replicates were sufficient to obtain averages within 5-10% of the best estimated mean, while 10-20% accuracy was obtained with 4-12 replicates for SiO₂ and >10 replicates for NH₄ ⁺ dark fluxes. Mean accuracy was considerably lower for all light incubations, probably due to the patchy distribution of the benthic microalgal community.     

Isolation of plasma membrane and binding of the Ca2+ antagonist nimodipine in Chlamydomonas reinhardtii

Chlorophyll-free plasma membranes of the unicellular green alga Chlamydomonas reinhardtii Dangeard were purified from a microsomal fraction using an aqueous polymer two-phase system of 6.5% (w/w) dextran T500, 6·5% (w/w) polyethylene glycol 3350, 60 mM NaCI, 0 33 M sucrose and 5 mM potassium phosphate (pH 7·8). The plasma membrane fraction contained only 2·4% of the microsomal membrane protein. Specific activity of the plasma membrane marker enzyme, K*, Mg²⁺-ATPase (EC 3.6.1.3). was enriched 9-fold over the microsomal fraction, and 22% of total activity was recovered in the upper, polyethylene glycol-rich phase. Contamination from intracellular membranes was minimal. K*, Mg²⁺-ATPase showed a pH optimum at about 6·5, and addition of 0·05% (w/v) Triton X-100 stimulated the activity 3-fold. [³H]-Nimodipinc was employed to characterize 1,4-dihydropyridine-specific membrane receptors. Two apparent binding sites with different affinities to nimodipine were found in the crude microsomal fraction. The separation of plasma membranes from intracellular membranes revealed that one binding site with higher affinity (K_D= 9 nM) was located on the plasma membrane and a second binding site with lower affinity (K_D= 36 nM) on an intracellular membrane The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments were comparable to those determined by equilibrium experiments. The maximum number of binding sites of the plasma membrane fraction and the intracellular membrane fraction was B_max= 440 and 470 fmol (mg protein)^-1, respectively. [³H]-Nimodipinc binding was inhibited by (±) verapamil and stimulated by D-cis-diltiazem in both fractions. Moreover, ethyle-neglycol-bis(2-aminoethylcther)-N, N'-tetraacctic acid (EGTA) inhibited [³H]-nimo-dipinc binding in the plasma membrane fraction but not in the intracellular membrane fraction This effect was cancelled by the addition of CaCl₂.     

Effects of dissolved oxygen concentration on lipase production by Rhizopus delemar

Summary The influence of the concentration of oxygen on lipase production by the fungus Rhizopus delemar was studied in different fermenters. The effect of oxygen limitation ( 47 mol/l) on lipase production by R. delemar is large as could be demonstrated in pellet and filamentous cultures. A model is proposed to describe the extent of oxygen limitation in pellet cultures. Model estimates indicate that oxygen is the limiting substrate in shake flask cultures and that an optimal inoculum size for oxygen-dependent processes can occur.Low oxygen concentrations greatly negatively affect the metabolism of R. delemar, which could be shown by cultivation in continuous cultures in filamentous growth form (D_optimal=0.086 h^-1). Continuous cultivations of R. delemar at constant, low-oxygen concentrations are a useful tool to scale down fermentation processes in cases where a transient or local oxygen limitation occurs.Symbols and Abbreviations CO Oxygen concentration in the gas phase at time = 0 (kg·m^-3) - C_{O 2i} Oxygen concentration at the pellet liquid interface (kg·m^-3) - C_{O 2i} Oxygen concentration in the bulk (kg·m^-3) - D Dilution rate (h^-1) - ID_{O 2} Diffusion coefficient for oxygen (m²·s^-1) - dw Dry weight of biomass (kg) - f Conversion factor (rs _{O 2} to oxygen consumption rate per m³) (-) - k Radial growth rate (m·s^-1) - K Constant - kla Volumetric mass transfer coefficient (s^-1) - klA Oxygen transfer rate (m^-3·s^-1) - kl Mass transfer coefficient (m·s^-1) - K _{O 2} Affinity constant for oxygen (mol·m^-3) - K _w Cotton plug resistance (m^-3·s^-1) - M Henry coefficient (-) - NV Number of pellets per volume (m^-3) - R Radius (m) - RO Radius of oxygen-deficient core (m) - RQ Respiration quotient (mol CO₂/mol O₂) - rs _{O 2} Specific oxygen consumption rate per dry weight biomass (kg O₂·s^-1[kg dw]^-1) - rX Biomass production rate (kg·m^-3·s^-1) - SG Soytone glucose medium (for shake flask experiments) - SG ₄ Soytone glucose medium (for tower fermenter and continuous culture experiments) - V Volume of medium (m^-3) - X Biomass (dry weight) concentration (kg·m^-3) - XR _o Biomass concentration within RO for a given X (kg·m^-3) - Y _{O 2} Biomass yield calculated on oxygen (kg dw/kg O₂) - Thiele modulus - Efficiency factor =1-(RO/R)³ (-) - Growth rate (m^-1·s^-1·kg^1/3) - Dry weight per volume of pellet (kg·m^-3)     

Determination of the inherent kinetics of immobilized glucose oxidase using a diffusion cell

The enzyme glucose oxidase (GO) was covalently immobilized onto a poly(vinyl alcohol) hydrogel, cross-linked with glutardialdehyde and a polyazonium salt. To compare the kinetic parameters of immobilized GO with the known kinetic parameters of soluble GO, the diffusion cell method was used.Between two compartments, containing solutions with different glucose concentrations, a GO-containing hydrogel membrane was placed. Simultaneous diffusion through and enzymatic reaction in the membrane occurred. In this way diffusional effects of the membrane could be eliminated from the effective kinetic parameters to yield the inherent kinetic parameters.It appeared that the enzymatic reaction is independent of the oxygen concentration at oxygen concentrations 0.22 mol m^–3 (Michaelis constant for oxygen < 0.22 mol m^–3). Further, the Michaelis constant for glucose does not change dramatically after immobilizing the enzyme. The maximal reaction rate is depending on the enzyme concentration. As the enzyme concentration in the membrane is not exactly known (mainly due to leakage of enzyme out of the membrane during membrane preparation), only an estimation of the turnover number can be made.The diffusion cell method is easy to carry out. Still, some recommendations can be made on the performance.List of Symbols _g , _0x partition coefficient of glucose and oxygen, respectively - thickness of the wetted membrane (m) - A _m surface area of membrane (m^–2) - C constant (mol² m^–3) - c _g , c _0x concentration of glucose and oxygen, respectively (mol m^–3) - c _g,0 c _g, glucose concentration at the filter-paper/membrane interface next to compartment A and B, respectively (mol m^–3) - c _{g, A} c _{g, B} glucose concentration in compartment A and B, respectively (mol m^–3) - c _GO glucose oxidase concentration (mol m^–3) - D _eff effective diffusion coefficient (m² s^–1) - D _m , D _sl diffusion coefficient in, respectively, the membrane and the solution layer (m² s^–1) - d _dl , d _df , d _sl thickness of, respectively, the diffusion layer, the filter-paper and the solution layer (m) - h _B initial slope of concentration versus time curve of compartment B (mol m^–3 s^–1) - J flux (mol m^–2 s^–1) - J ₀ flux in the membrane at membrane/filter-paper interface next to compartment A and B, respectively (mol m^–2 s^–1) - J _A , J _B flux leaving compartment A and entering compartment B, respectively (mol m^–2 s^–1) - J _m flux through the membrane (mol m^–2 s^–1) - k total mass transfer coefficient (m s^–1) - k ₁ , k ₂ rate constant of a particular reaction step (m³ mol^–1 s^–1) - k_–1, k_–2 rate constant of a particular reaction step (s^–1) - k _cat (intrinsic) catalytic constant of turnover number (s^–1) - k _cat ^* inherent catalytic constant, determined by inserting D _m (s^–1) - k _cat ^** inherent catalytic constant, determined by inserting D _eff (s^–1) - k _m (g) (intrinsic) Michaelis constant for glucose (mol m^–3) - k _m (o) (intrinsic) Michaelis constant for oxygen (mol m^–3) - k _m ^* (g) inherent Michaelis constant for glucose (mol m^–3) - k _m ^* (o) inherent Michaelis constant for oxygen (mol m^–3) - m _GO number of moles of GO present (mol) - P _m permeability of glucose in the mebrane (m s^–1) - P _eff effective permeability (m s^–1) - V volume (m³) - v ₀ initial reaction velocity (mol m^–3 s^–1) - V _max ^** inherent maximal reaction velocity, determined by inserting D_eff (mol m^–3 s^–1) - x distance (m)     

Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: Role of Ca2+-dependent K+ channels

Summary Eleven different secosteroids or steroids (10^–10 to 10^–8 m) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibianNecturus kidney while recording basolateral cell membrane potentialV _m. Seven of these molecules (1,25(OH)₂D₃, 25(OH)D₃, 24,25(OH)₂D₃, 5,6-trans-25(OH)D₃, 19-diol-cholesterol, estradiol and testosterone) resulted in changes ofV _m (V _m) occurring in a few seconds, the largest V _m being observed with 1,25(OH)₂D₃, +6.5±0.75 mV (n=19); these seven (seco)steroids but not the four inactive sterols (vitamin D₃, cholesterol, 1D₃ and aldosterone) possess a hydroxyl group on at least one carbon of the C₁₇ to C₂₅ lateral chain of the sterol ring. The V _m effect was present in Na⁺-free or Cl^–-free media, but it was abolished in HCO₃-free media. Depolarization of cell membrane potential by addition of glucose, 11mm, in luminal perfusion fluid abolished the 1,25(OH)₂D₃-evoked V _m effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K⁺ conductances, suppressed the 1,25(OH)₂D₃-evoked V _m effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca²⁺-dependent K⁺ channels also abolished the 1,25(OH)₂D₃-evoked depolarization. Such observations are consistent with the presence of Ca²⁺-dependent K⁺ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)₂D₃ and probably by other (seco)steroids.     

Radiation Chemistry of Foods

An intermediate radical, ?H₂OH, was produced in aqueous methanol solution containing nitrous oxide by γ-irradiation. Yields of ethylene glycol and formaldehyde, the major and the minor product from ?H₂OH, respectively, changed on the addition of some solutes. Cysteine lowered the both product yields to zero even at a low concentration of 5 × 10^?5m. Oxygen of low concentrations (2.5~7.5 × 10^?5 m) changed effectively the major product from ethylene glycol to formaldehyde. k _(CySH+?H2OH)/k_(O2+?H2OH) was calculated as 0.5.

Ascorbic acid (5 × 10^?5 m) lowered ethylene glycol yield to 48%, cystine (10^?3m) to 15%, methionine (10^?3m) to 31%, histidine (10^?3m) to 42%, tryptophan (10^?3m) 46%, tyrosine (10^?3m) to 77%, phenylalanine (10^?3m) to 73%, hypoxanthine (10^?3m) to 37%, adenine (10^?3m) to 52%, uracil (10^?3m) to 20%, thymine (10^?3m) to 10%, cytosine (10^?3 m) to 49%, rutin (10^?3m) to 23%, pyrogallol (10^?3m) to 41%, and gallic acid (10^?3m) to 78% of the control. These results suggest that the reactions of the secondary radicals such as ?H₂OH perform an important role in material change of foods irradiated with γ rays.