S-nitrosoglutathione incorporated in poly(ethylene glycol) matrix: potential use for topical nitric oxide delivery.

Incorporation of nitric oxide (NO) donors in non-toxic polymeric matrices can be a useful strategy for allowing topical NO delivery. We have incorporated the NO-donor S-nitrosoglutathione (GSNO) into a liquid poly(ethylene glycol) (PEG)/H2O matrix through the S-nitrosation of GSH by a NO/O2 gas mixture. Kinetic measurements of GSNO decomposition associated with NO release were performed at 25, 35, and 45 degrees C in the dark and under irradiation with UV/Vis light, lambda>480 nm and lambda=333 nm. NO release from the liquid matrix to the gas phase was confirmed by mass spectrometry. The PEG/H2O matrix stabilizes GSNO leading to expressive reductions in the initial rates of thermal and photochemical NO release, compared to aqueous GSNO solution. This matrix effect is assigned to diffusional constrains imposed on the escape of the NO and GS radicals formed in the solvent cage. This effect allows the storage of PEG-GSNO formulations for extended periods (more than 65 days at freezer) with negligible decomposition. PEG-GSNO formulation seems therefore to be applicable in topical NO delivery and GSNO displays potential as a percutaneous absorption enhancer. Moreover, the rate of NO release can be locally increased by irradiation with visible light.     

Toxic effects of some alcohol and ethylene glycol derivatives on Cladosporium resinae.

Eleven commercially available alcohol and ethylene glycol derivatives were tested for their toxicity toward a problem organism in jet fuel, Cladosporium resinae. In the presence of glucose, 20% (vol/vol) ethylene glycol monomethyl ether prevented spore germination and mycelial growth, and 10% (vol/vol) 2-ethoxybutanol, 10% 2-isopropoxyethanol, 10% 3-methoxybutanol, 5% 2-butyloxyethanol, 5% ethylene glycol dibutyl ether, and 5% diethylene glycol monobutyl ether were found to have similar effects. In a biphasic kerosene-water system, 3-methoxybutanol, 2-butyloxyethanol, and diethylene glycol monobutyl ether were again found to be more toxic than ethylene glycol monomethyl ether. Considerable potassium efflux, protein leakage, and inhibition of endogenous respiration were observed in the presence of the more toxic compounds. 2-Butyloxyethanol also caused loss of sterols from cells.     

Interaction between dry starch and plasticisers glycerol or ethylene glycol, measured by differential scanning calorimetry and solid state NMR spectroscopy

The interaction of crystalline amylose and of crystalline and amorphous amylopectin with the plasticisers glycerol or ethylene glycol in the absence of water was studied, by using differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Upon heating starch freshly mixed with plasticisers, a strong exothermal interaction enthalpy of ΔH−35 J/g was detected by DSC. At room temperature glycerol interacts mainly with the amorphous starch regions, the interaction taking 8 days to reach equilibrium. For ethylene glycol the interaction is faster, taking four days to reach equilibrium, and the rate is not affected by crystallinity. Ethylene glycol interacts in a more ordered manner with amorphous than with crystalline material, resulting in a narrower ethylene glycol cross-polarisation magic angle spinning (CP/MAS) signal when equilibrium is reached at room temperature. Upon heating, more glycerol or ethylene glycol is immobilised, but in a less ordered manner than upon storage at room temperature. This results in a more intense, but broader plasticiser CP/MAS signal upon heating. Interaction in a more ordered manner probably implies interaction with more of the hydroxy groups of the plasticiser. The polysaccharide mobility is increased more when the plasticiser interacts in a more ordered manner, as observed by small starch signals in HP/DEC spectra.     

Osmotic characteristics of sheep and cattle embryos

Ten minutes of exposure to increasing concentrations of sucrose caused a proportional decrease in the volume of sheep late morulae, their relative volume changed as a linear function of the reciprocal of the osmolality of the medium. Day 6 sheep and Day 7 cattle embryos responded to the addition of permeating cryoprotectants by an initial shrinkage which was followed by gradual reexpansion. After 1.25 min exposure the relative volumes of sheep and cattle embryos respectively were 20 and 25% smaller in glycerol than in ethylene glycol. The volumes of cattle and sheep embryos remained smaller in glycerol than in ethylene glycol up to the final observation at 30 min. The osmotic response of sheep late morulae to 2.0 M propylene glycol was intermediate between their response to 2.0 M glycerol and to 2.0 M ethylene glycol. These results indicate that Day 6 sheep and Day 7 cattle embryos are more permeable to ethylene glycol than to glycerol.     

Dissociation and reconstitution of a DNA polymerase alpha-primase complex

The conditions for dissociation of the DNA polymerase alpha-primase complex (DNA polymerase alpha 1) have been examined. It was revealed that 50% ethylene glycol effectively dissociated the complex. The dissociated DNA polymerase and primase were purified to eliminate cross-contaminating activities by column chromatography using buffers containing 50% ethylene glycol. The sedimentation coefficients of the purified DNA polymerase and primase were 7.1S and 5.7S, respectively. These two enzymes were mixed in the presence of 20% ethylene glycol and the mixture was sedimented through a glycerol gradient containing no ethylene glycol. The DNA polymerase and primase activities co-sedimented at 9.1S which corresponds to the S value of intact alpha 1, indicating the reconstitution of the DNA polymerase alpha-primase complex.     

Liquid-liquid partitioning of some enzymes, especially phosphofuctokinase, from Saccharomyces cerevisiae at subzero temperature

The effects of low temperature (−18°C) on the stability and partitioning of some glycolytic enzymes within an aqueous two-phase system were studied. The enzymes were phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase present in a crude extract of bakers' yeast. The partitioning of pure phosphofructokinase, isolated from bakers' yeast, was also examined. The two-phase systems were composed of water, poly(ethylene glycol), dextran, and ethylene glycol and buffer. The influence on the partitioning of the presence of ethylene glycol, phenylmethylsulfonyl fluoride and poly(ethylene glycol)-bound Cibacron Blue F3G-A was investigated at −18, 0 and (in some cases) 20°C. The presence of ethylene glycol, phase polymers and low temperature stabilized all three enzyme activities. Cibacron Blue, an affinity ligand for phosphofructokinase, increased its partitioning into the upper phase with decreasing temperature. Depending on the conditions, various amounts of the enzymes were recovered at the interface, also in systems not containing ethylene glycol. The implications of the observed effects on the use of aqueous two-phase systems for the extraction and fractionation of proteins are discussed.     

Adsorption of lipoproteins with the aid of carboxymethylchitosan mircrospheres crosslinked with poly(ethylene glycol) bisglycidyl ether

Carboxymethylchitosan microspheres crosslinked with poly(ethylene glycol) bisglycidyl ether were prepared and then tested as an adsorbent for selective removal of low-density lipoprotein (LDL) in human plasma. The microspheres were formed by a method of electrostatic instillation and crosslinked with poly(ethylene glycol) bisglycidyl ether. FTIR spectral analyses and X-ray photoelectron spectroscopy revealed that carboxymethylchitosan was crosslinked through amino groups to poly(ethylene glycol) bisglycidyl ether. The plasma lipoprotein sorption tests showed that the adsorption properties of the crosslinked microspheres for LDL were dependent on the concentrations of carboxymethylchitosan and poly(ethylene glycol) bisglycidyl ether. When the concentrations of carboxymethylchitosan and poly(ethylene glycol) bisglycidyl ether were 3.5% and 6%, respectively, 40% LDL and lower than 10% high density lipoprotein in plasma could be removed and the adsorption could be reach an equilibrium in 30 min.     

In vitro assessment of a direct transfer vitrification procedure for bovine embryos

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.     

A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability

Mouse morulae were exposed to solutions containing 30-50% of permeable agents (ethylene glycol, glycerol, propylene glycol) in modified phosphate-buffered saline (PB1 medium) at 20 degrees C for 20 min. A high percentage of them developed to expanded blastocysts in culture, after exposure to 30% and 40% ethylene glycol (98 and 84%, respectively), or 30% glycerol (88%). Ethylene glycol and glycerol were diluted to 30 and 40% with PB1 medium or with PB1 containing 30% Ficoll or 30% Ficoll + 0.5 M-sucrose, immersed in liquid nitrogen in straws and warmed in 20 degrees C water. Solutions containing 40% of a permeable agent with Ficoll did not crystallize during cooling or warming. Mouse morulae were exposed to 40% ethylene glycol in PB1 medium containing 30% Ficoll (EF) or PB1 medium + 30% Ficoll + 0.5 M-sucrose (EFS) for 5-20 min at 20 degrees C. EFS solution was non-toxic to the embryos during 5 min of exposure. When embryos, equilibrated in EFS solution for 2 or 5 min at 20 degrees C, were vitrified at -196 degrees C and were warmed rapidly, nearly all embryos developed in culture (97-98%), and 51% developed to live young at term after transfer. This method, which results in virtually no decrease in embryonic viability, may be of practical use for embryo preservation.     

Relationship Between Ethylene Release,Membrane Permeability and Activity of Polyphenol Oxidase Changes of Duck (Pears Pyrus bretschnideri Rehder) During Low Temperature Storage

Duck pear (Pyrus bretschneideri Rehder) tends to develop browning core after 55 to 60 days storage at low temperature (0℃). Following physiological changes of the duck pear during storage at different temperature were investigated: (1) As compared with 20℃, ethylene release is obviously decreased and its peak is retarded for 15 days at 0℃. Levels of internal ethylene are variant at different individuals harvested at same time. Concentrations of internal ethylene are in accord with ethylene release. The higher internal ethylene is, the easier the pear core becomes brown. (2) At 0℃, activity of polyphenol oxidase in the core increases with ethylene release enhancement. After ethylene peak passes, its activity is lower than before. (3) The electric conductivity of cores is higher at 0℃ than at 20℃. During post climacteric period, the electric conductivity increases irreversibly, then browning core occurs. From above results, it is concluded that interactions between two factors induce the browrang core of the duck pears at low temperature. One is chilling injury caused by low temperature, another is ethylene function. They stimulate the activity of polyphenol oxidase and enhance the membrane permeability.     

Clathrin-Dependent and Clathrin-Independent Endocytosis are Differentially Sensitive to Insertion of Poly (Ethylene Glycol)-Derivatized Cholesterol in the Plasma Membrane

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036–3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.     

Design, synthesis, and biocompatibility of an arginine-based polyester

Polycations are very useful in biotechnology. However, most existing polycations have high toxicity that significantly limits their clinical translation. We designed poly(ethylene argininylaspartate diglyceride) (PEAD) that is based on arginine, aspartic acid, glycerol, and ethylene glycol. A set of in vitro assays demonstrated that PEAD exhibited no cytotoxicity at 1 mg/mL, which is at least 100 times higher than the widely used polycation-polyethylenimine. Subcutaneous injection of 1 mg PEAD in rats did not cause an adverse response acutely or after 4 weeks. Zeta potential measurements revealed that PEAD has high affinity to biological polyanions such as DNA and hyaluronic acid. This polycation represents a new platform of biocompatible polycations that may lead to clinical innovations in gene therapy, controlled release, tissue engineering, biosensors, and medical devices.     

Development of a sensitive radiorespiration method for detecting microbial activity at subzero temperatures

We have developed a simple and sensitive method to detect microbial respiration at subzero temperatures. Microbial activity was detected by measuring (14)CO(2) evolved during the microbial-mediated mineralization of [1-(14)C] acetic acid or [2-(14)C] glucose in microcosm assays using modified (14)CO(2) traps. Various (14)CO(2) traps, designed to withstand freezing at subzero temperatures, were tested for their quench characteristics during liquid scintillation spectrometry and their ability to trap (14)CO(2). Solutions consisting of 1 M KOH supplemented with 20% or 30% v/v ethylene glycol did not freeze at temperatures above -20 degrees C and had a minor quenching effect on liquid scintillation spectrometry. Addition of ethylene glycol did have an effect on the efficiency of (14)CO(2) trapping, as the cumulative recovery of (14)CO(2) was reduced by 14% and 32% in the 1 M KOH+20% ethylene glycol and 1 M KOH+30% ethylene glycol solutions, respectively. Using the modified (14)CO(2) traps, microbial activity in representative Canadian high Arctic environmental samples was detected at temperatures as low as -15 degrees C. This simple method allows for sensitive, specific, and reliable detection of microbial activity occurring at subzero temperatures and is readily adaptable for studies in other cryoenvironments.     

In vitro evaluation of ram sperm frozen with glycerol, ethylene glycol or acetamide

The aim was to assess the in vitro effect of glycerol, ethylene glycol or acetamide on frozen-thawed ram spermatozoa. Aliquots of each sixteen ejaculates from four rams of the Morada Nova breed were diluted in Tris-egg yolk with glycerol (5%), ethylene glycol (3% or 5%) or acetamide (3% or 5%) and frozen at -196°C. After thawing, progressive sperm motility was greater (P<0.05) in cryopreservation with glycerol 5% and ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Acrosome integrity was greater (P<0.05) with ethylene glycol 5% than acetamide (3% or 5%). The percentage of sperm without oxidative stress was greater (P<0.05) with ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Plasma membrane integrity was greater with glycerol 5% (P<0.05) than with the other cryoprotectants. Thus, it is concluded that glycerol 5% and ethylene glycol 3% or 5% protect ram sperm against the harmful effects of freezing and that glycerol 5% offers greater protection to sperm plasma membrane.     

Characterization of DNA polymerase alpha activity from a mouse DNA temperature-sensitive mutant, strain tsFT20, which shows a defect in DNA polymerase alpha activity at restrictive temperatures

tsFT20 cells derived from mouse FM3A cells are DNA temperature-sensitive mutants, which have heat-labile DNA polymerase alpha activity. When tsFT20 cells were incubated at restrictive temperatures, intracellular levels of DNA polymerase alpha activity changed biphasically, showing an initial fast decrease (phase I) and a subsequent slow decrease (phase II). The activity of DNA polymerase alpha from tsFT20 cells cultured at a permissive temperature (33 degrees C) was greatly increased by the addition of glycerol or ethylene glycol to the reaction mixture, while little increase in enzyme activity was observed at any concentration of glycerol or ethylene glycol tested with the enzyme from the cells cultured at a restrictive temperature (39 degrees C) for 8 h (phase II). The activity of DNA polymerase alpha from wild-type cells was also increased by the addition of glycerol but the increase was much less than that in the tsFT20 cells. An in vitro preincubation experiment showed that DNA polymerase alpha from tsFT20 cells cultured at 33 degrees C very rapidly lost its ability to be stimulated by glycerol. Furthermore, the experiment using the extracts prepared from tsFT20 cells cultured at 39 degrees C for various periods showed that the ability to be stimulated by glycerol decreased with the duration of incubation time at 39 degrees C. DNA polymerase alpha from the revertants, which can grow at 39 degrees C and exhibit a partial recovery in heat stability of DNA polymerase alpha activity, showed an intermediate response to glycerol, between those of DNA polymerase alpha from tsFT20 and from the wild-type cells. Finally, it was observed that the level of enzyme activity that can be stimulated by glycerol correlated well with the DNA synthesizing ability of tsFT20 cells.     

Cryoprotectants for Crithidia fasciculata Stored at -20 C, with Notes on Trypanosoma gambiense and T. conorhini

SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log-phase or end-of-log-phase cultures were frozen for 24–48 hr at ∼ -20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2'-dioxyethanol (diethylene glycol); 1,2,4-butanetriol; 1,4-cyclohexanediol; dimethylsulfoxide; propylene glycol; and N -acetylethanolamine. Several sugars were active, e.g., D-arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood-enriched media + 2-2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3-week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.     

Enzymic acylation of 6-aminopenicillanic acid

The enzymic synthesis of benzylpenicillin from 6-aminopenicillanic acid in the presence of poly (ethylene glycol) has been studied. With equimolar initial concentrations (20 mM) of 6-aminopenicillanic acid and phenylacetic acid a 60% conversion to benzylpenicillin can be achieved at 10°C and pH 5.2 in the presence of 45% (w/v) poly(ethylene glycol). Under these conditions the lactam ring of the benzylpenicillin and 6-aminopenicillanic acid and the enzyme, penicillin acylase (penicillin amidase, penicillin amidohydrolase, EC 3.5.1.11), were more stable than in the absence of the polyol.     

Estimation of tryptophyl and tyrosyl exposure in tryptophan-rich proteins by ultraviolet difference spectrophotometry. Lysozyme and Chymotrypsinogen.

Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophanamide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (delta epsilon) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287 nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because delta epsilon285 was larger than delta epsilon292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins. The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more beta-structured for chymotrypsinogen.     

Biodegradation of Ethylene Glycol by a Salt-Requiring Bacterium

A gram-negative nonmotile rod which was capable of using 1,2-(14)C-ethylene glycol as a sole carbon source for growth was isolated from a brine pond, Great Salt Lake, Utah. The bacterium (ATCC 27042) required at least 0.85% NaCl for growth and, although the chloride ion was replaceable by sulfate ion, the sodium ion was not replaceable by potassium ion. The maximal concentration of salt tolerated for growth was approximately 12%. The bacterium was oxidase-negative when N,N-dimethyl-p-phenylenediamine was used and weakly positive when N,N,N',N'-tetramethyl-p-phenylenediamine was used. It grows on many sugars but does not ferment them, it does not have an exogenous vitamin requirement, and it possesses a guanine plus cytosine ratio of 64.3%. Incorporation of ethylene glycol carbon into cell and respired CO(2) was quantitated by use of radioactive ethylene glycol and a force-aerated fermentor. Glucose suppressed ethylene glycol metabolism. Cells grown on ethylene and propylene glycol respired ethylene glycol in a Warburg respirometer more rapidly than cells grown on glucose. Spectrophotometric evidence was obtained for oxidation of glycolate to glyoxylate by a dialyzed cell extract.     

The effect of ethylene glycol on the phytovolatilization of 1,4-dioxane

Phytoremediation at contaminated sites is often complicated by the presence of more than one chemical However, the effects of common co-contaminants such as ethylene glycol on the phytoremediation of other chemicals, e.g., 1,4-dioxane, is not well understood. Field studies with DN34 poplar trees revealed a 28% decline in growth rate in response to 10 g/L ethylene glycol in the groundwater, thus indicating a significant and deleterious effect on tree viability, and likely, the plants' utility for phytoremediation. Thorough investigations using Arabidopsis thaliana, with its small size and rapid life cycle, indicated significant growth reduction at 10 g/L and complete inhibition of germination at 40 g/L ethylene glycol Ethylene glycol was almost as severe a stressor as the well characterized osmotic inhibitor, sorbitoL Watering potted trees with 10 g/L ethylene glycol reduced their growth by more than 50%, and similar results were observed in hydroponically grown poplar and willow trees. Under hydroponic conditions, 60 g/L ethylene glycol inhibited the phytovolatilization of l,4-dioxane by more than 80%, and all trees evapo-transpired 1,4-dioxane less efficiently than water. In fact, this efficiency differed between trees and the difference became more pronounced in the presence of ethylene glycol.