Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli.

Spontaneous mutants of Escherichia coli able to grow on ethylene glycol as a sole source of carbon and energy were obtained from mutants that could grow on propylene glycol. Attempts to obtain ethylene glycol-utilizing mutants from wild-type E. coli were unsuccessful. The two major characteristics of the ethylene glycol-utilizing mutants were (i) increased activities of propanediol oxidoreductase, an enzyme present in the parental strain (a propylene glycol-positive strain), which also converted ethylene glycol into glycolaldehyde; and (ii) constitutive synthesis of high activities of glycolaldehyde dehydrogenase, which converted glycolaldehyde to glycolate. Glycolate was metabolized via the glycolate pathway, which was present in the wild-type cells; this was indicated by the induction in ethylene glycol-grown cells of glycolate oxidase, the first enzyme in the pathway. Glycolaldehyde dehydrogenase was partially characterized as an enzyme of this new metabolic pathway in E. coli, and glycolate was identified as the product of the reaction. This enzyme used NAD and NADP as coenzymes, although the NADP-dependent activity was about 10 times lower than the NAD-dependent activity. Uptake of [14C]ethylene glycol was dependent on the presence of the enzymes capable of metabolism of ethylene glycol. Glycolaldehyde and glycolate were identified as intermediate metabolites in the pathway.     

乙二醇对木瓜蛋白酶催化活性的影响

对不同浓度乙二醇水溶液影响木瓜蛋白酶水解酪蛋白的催化活性及构象的改变进行研究。结果表明,木瓜蛋白酶在乙二醇水溶液中水解酪蛋白的活性显著下降。动力学测定表明,木瓜蛋白酶在乙二醇水溶液中最大反应速度明显下降;差示光谱显示,在乙二醇水溶液中木瓜蛋白酶的二级结构发生了变化;荧光发射光谱显示,木瓜蛋白酶在乙二醇水溶液中发射峰位几乎没移动,但发射强度明显增高。     

Synthesis and bioactivities of poly(ethylene glycol)–chitosan hybrids

Poly(ethylene glycol)–chitosan hybrids of various molecular weights having different degree of substitution were synthesized, by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde, to study their bioactivities. The influence of these chitosan derivatives on the reactive oxygen species generation from canine polymorphonuclear leukocyte cells was investigated in vitro by chemiluminescence response. Reactive oxygen species generation by the influence of poly(ethylene glycol)–chitosan hybrids was decreased with the increase of degree of substitution. The reduction of interaction of poly(ethylene glycol)–chitosan hybrids with polymorphonuclear leukocyte cells might be caused by the decrease of amino group in chitosan main chain and increase of the steric hindrance by poly(ethylene glycol) chain. The influence of the poly(ethylene glycol)–chitosan hybrids on complement component C3 activation was investigated by single radial immunodiffusion method. Influence on complement component C3 activation by poly(ethylene glycol)–chitosan hybrids was almost same as chitosan.     

Enzyme action in polymer and salt solutions. I. Stability of penicillin acylase in poly(ethylene glycol) and potassium phosphate solutions in relation to water activity

The stability of penicillin acylase (penicillin aminohydrolase, EC 3.5.1.11) was studied in poly(ethylene glycol) and potassium phosphate solutions. Enzyme stability measured as the half-life of the enzymatic activity and the transition temperature determined by differential scanning calorimetry, correlated well. The enzyme stability could not be related to the water activity as a measure of solute-solvent interaction. It seems to be related more to the concentration of the solutes and much less to the molecular weight of poly(ethylene glycol). The stabilizing effect of poly(ethylene glycol) is also discussed in terms of poly(ethylene glycol)-protein interactions.     

Generation of selective microenvironment at the cell periphery through bulk-phase hydrophilic polymers

In order to understand the previously demonstrated effect of poly(ethylene glycol) on the stimulation of lymphocyte responses to syngeneic tumor cells (Ben-Sasson, S.A. and Henkart, P.A. (1977) J. Immunol. 119, 227–231), we have investigated the effects of addition of poly(ethylene glycol) to the medium in a number of cellular systems. The binding of trimeric IgG to tumor-lymphocyte Fc receptors was greatly enhanced by poly(ethylene glycol); a substantial increase in binding of trimeric IgG to non-Fc-receptor-bearing tumor cells was also observed. Similarly, the binding of labeled bovine serum albumin to lymphocyte surfaces was increased by poly(ethylene glycol), implying that nonspecific binding of proteins to cells was generally enhanced. The dose-response curve of concanavalin A mitogenesis was shifted to the right, as would be expected from a local increase in concanavalin A concentration. Antibody binding to erythrocytes as detected by complement lysis was similarly increased. It was found that in aqueous two-phase mixtures created by poly(ethylene glycol) and dextran, erythrocytes partition into the dextran phase through exclusion into dextran-rich microdroplets. It is proposed that addition of poly(ethylene glycol) to cell culture media creates a similar separate phase around the cell surface in which the local concentration of proteins is greater than that in the bulk medium. This concept explains many of the diverse effects of addition of poly(ethylene glycol) to the medium. It also can partially explain the requirement for serum to observe the poly(ethylene glycol) effect on the lymphocyte response to syngeneic tumor cells.     

Decrease of transport of some polyols in sickle cells

This paper reports the results of kinetic studies on the inward net-flux of small non-electrolytes (ethylene glycol, glycerol and erythritol) in sickle cells as compared to normal erythrocytes. Net transport rates were evaluated by turbidimetric measurements for ethylene glycol and glycerol and by hematocrit monitoring for erythritol. A 2-fold and 4-fold reduction in the permeability coefficient for ethylene glycol and glycerol, respectively, were found in sickle cells as compared to normal erythrocytes. In contrast, no significant changes in erythritol transport kinetics were observed. The dependence of glycerol permeability on temperature, pH and oxygenation is the same in both types of cells. A significant correlation was observed between glycerol permeability and cell density only for sickle cells. The results indicate that irreversible modifications of membrane proteins, responsible for the glycerol and ethylene glycol transport, do occur in sickle cells.     

一株乙二醇氧化酶菌株的筛选及催化特性的初步研究

利用富集培养技术从土壤中筛选获得1株高活性二醇氧化活性菌株Brevibacterium sp.CCZU12-1。以Brevibacterium sp.CCZU12-1静息细胞为催化剂,最适催化反应温度、反应pH和金属离子添加量分别为30℃、6.5和Mn2＋0.1 mmol/L。在最佳条件下,转化200 mmol/L乙二醇24 h,羟基乙酸的产率为94.6%,分批补料乙二醇5批,羟基乙酸的累积浓度为972 mmol/L。     

Physicochemical and biological properties of poly(ethylene glycol)-coupled immunoglobulin G

In order to develop a new intravenous immunoglobulin G (IgG), IgG was covalently coupled to poly(ethylene glycol) previously activated by cyanuric chloride. The poly(ethylene glycol) coupled IgG obtained was studied for physicochemical and biological properties such as molecular structure, size-exclusion chromatographic behaviour, surface activity, interfacial aggregability, heat aggregability inducing nonspecific complement activation, and antigen-binding activity. The poly(ethylene glycol) coupling to IgG increased the apparent Stokes' radius and the surface activity of IgG and stabilized IgG on heating and/or on exposure to interface, while no structural denaturation of IgG was observed. The suppressed nonspecific aggregability was interpreted mainly by difficulty in association between the modified IgG molecules. These results indicated the use of the poly(ethylene glycol)-coupled IgG as an intravenous preparation and also as an additive stabilizing intact IgG for intravenous use.     

Modification of the contractile properties of rabbit skeletal muscle by ethylene glycol

The effect of ethylene glycol on the contractile properties of skeletal muscles was studied using glycerinated rabbit psoas muscle fibers. Measurements were made at an ionic strength of 0.2 M, pH 7.0, and at 10 degrees C. Ethylene glycol reversibly reduced isometric tension, active stiffness, the tension-to-stiffness ratio, and the shortening velocity at zero load (Vo) in a dose-dependent fashion. Ethylene glycol also reduced the Ca sensitivity for contraction. The extent of the reduction in Vo by ethylene glycol was much larger than that in the actomyosin ATPase activity reported by Travers and Hillaire (Eur. J. Biochem. 98, 293-299 [1979]). Although ethylene glycol reduced tension and Vo, the MgATP concentration dependence of these two quantities was almost unaffected. These results suggest that in the presence of ethylene glycol, force produced by crossbridges in the principal force-producing state is reduced and/or the relative population of the attached crossbridges in the low-force state increases. The results also suggest that the reduction in Vo by ethylene glycol is caused not only by a reduction in the actomyosin ATPase activity but also by a reduction in the shortening distance per mole of ATP split.     

Glycerol and ethylene glycol: members of a new class of repellents of Escherichia coli chemotaxis

By using the chemical-in-plug method, we found that glycerol and ethylene glycol caused negative chemotaxis in wild-type cells of Escherichia coli; the threshold concentration was about 10(-3) M for both chemicals. As with other known repellents, the addition of glycerol or ethylene glycol induced a brief tumble response in wild-type cells but not in generally nonchemotactic mutants. Experiments with mutants defective in various methyl-accepting chemotaxis proteins (MCPs) revealed that the presence of any one of three kinds of MCPs (MCP I, MCP II, or MCP III) was necessary to give a tumble response to these repellents. Consistently, it was found that the methylation-demethylation system of MCPs was involved in the adaptation of the cells to these repellents. The effect of glycerol or ethylene glycol was not enhanced by lowering the pH of the medium, and glycerol did not alter the membrane potential of the cells. All of these results suggest that glycerol and ethylene glycol are members of a new class of repellents which produce a tumble response in the cells by perturbing the MCPs in the membrane.     

Effect of poly(ethylene glycol) on the polarity of aqueous solutions and on the structure of vesicle membranes

The partitioning of TEMPO into phosphatidylcholine vesicle membranes is reduced upon addition of poly(ethylene glycol). This is caused by reduced polarity of the aqueous phase as well as decreased membrane fluidity in the presence of poly(ethylene glycol). The isotropic hyperfine splitting of TEMPO in aqueous poly(ethylene glycol) solutions was used as a measure of solvent polarity. The alterations of the membrane fluidity were detected by means of two different fatty acid spin labels. The influences of physicochemical properties of an aqueous poly(ethylene glycol) phase on the membrane structure of cells and vesicles are discussed in the light of membrane fusion.     

Immobilized metal ion affinity partitioning of cells in aqueous two-phase systems: erythrocytes as a model

Immobilized metal ion affinity partitioning of erythrocytes from different species is described. We have explored the affinity between transition metal chelates and metal-binding sites situated on the cell surface by partitioning in aqueous two-phase system composed of poly(ethylene glycol) and dextran. Soluble metal-chelate-poly(ethylene glycol) was prepared by fixing metal ions to poly(ethylene glycol) via the covalently bonded chelator, iminodiacetic acid. The partitioning behaviour of erythrocytes in systems at different concentrations of the ligand was tested. The copper-chelate-poly(ethylene glycol) was quite effective in the affinity extraction of human and rabbit erythrocytes, while the zinc-chelate-poly(ethylene glycol) displayed significant affinity only to the rabbit cells. Furthermore, the influence of various effectors such as imidazole, sialic acid on immobilized metal ion affinity partitioning of erythrocytes was examined.     

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.     

Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.     

Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10–14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.Abbreviations EG ethylene glycol - DiEG diethylene glycol - TriEG triethylene glycol - TeEG tetraethylene glycol - PEG polyethylene glycol (molecular mass indicated)     

Glycolic acid production using ethylene glycol-oxidizing microorganisms.

Screening for microorganisms oxidizing ethylene glycol to glycolic acid was carried out. Among stock cultures, several yeasts and acetic acid bacteria showed high glycolic acid producing activity. Pichia naganishii AKU 4267 formed the highest concentration of glycolic acid, 35.3 g/l, from 10% (v/v) ethylene glycol (molar conversion yield, 26.0%). Among soil isolates, Rhodotorula sp. 3Pr-126, isolated using propylene glycol as a sole carbon source, formed the highest concentration of glycolic acid, 25.1 g/l, from 10% (v/v) ethylene glycol (molar conversion yield, 18.5%). Rhodotorula sp. 3Pr-126 showed higher activity toward 20% (v/v) ethylene glycol than P. naganishii AKU 4267. Optimization of the conditions for glycolic acid production was investigated using P. naganishii AKU 4267 and Rhodotorula sp. 3Pr-126. Under the optimized conditions, P. naganishii AKU 4267 and Rhodotorula sp. 3Pr-126 formed 105 and 110 g/l of glycolic acid (corrected molar conversion yields, 88.0 and 92.2%) during 120 h of reaction, respectively.     

The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase.

The influence of temperature on cytochrome c oxidase (CCO) catalytic activity was studied in the temperature range 240-308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcal x mol-1; the molecular activity or turnover number was 12 s-1 at 280 K in the absence of ethylene glycol. CCO activity was studied between 240 and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal x mol-1; the molecular activity was 1 s-1 at 280 K. Ethylene glycol inhibits CCO by lowering the activity of water. The rate limitation in electron transfer (ET) was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash-induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s-1, 150 s-1 and 30 s-1 and these dropped to 220 s-1, 27 s-1 and 3 s-1 at 240 K. The activation energies were 5 kcal.mol-1, 7 kcal.mol-1, and 8 kcal.mol-1, respectively. The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cytochrome a oxidation and the slowest rate to the re-reduction of cytochrome a followed by its oxidation. There are two comparisons that are important: (a). with vs. without ethylene glycol and (b). steady state vs. flash-induced oxidation. When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle. In the present work that would mean after a flash-induced oxidation and the start of the next reduction/oxidation cycle.     

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces to biological hydrogels for rapid and efficient cell micropatterning

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.     

Relationship Between Solute Permeability and Osmotic Remediability in a Galactose-Negative Strain of Saccharomyces cerevisiae

An osmotic remedial allele, gal 7-1, in the galactose pathway of Saccharomyces cerevisiae responds to either penetrating (ethylene glycol and diethylene glycol) or nonpenetrating (KCl, NaCl, and sorbitol) solutes in the growth medium. Extracts from cells grown under restrictive conditions gave no increase in enzyme activity (gal-1-phosphate, uridylyl transferase) when exposed to the penetrating solutes; thus protein synthesis or possibly polymer assembly is proposed as the critical step remedied by the addition of the solutes.