Reactive products formed by peroxidase catalyzed oxidation of p-phenetidine

The drug 4-nitroquinoline 1-oxide (4NQO) is a potent inhibitor of Dictyostelium discoideum spore germination. This inexpensive, water soluble drug is active at a concentration of 5 micrograms/ml (26 microM) and permeates the spore at all stages in germination. Spores subjected to 4NQO treatment exhibit an irreversible blockage of myxamoebae emergence, but spore activation, post-activation lag, and swelling are not affected. Swollen 4NQO-treated spores lose the outer two spore walls but lack the ability to degrade the innermost wall. The drug does not affect oxygen uptake during post-activation lag or swelling, and only a stage specific depression in O2 uptake is observed when control spores begin to release myxamoebae. When added early in germination, 4NQO blocks the incorporation of [3H] uracil into a cold trichloroacetic acid (TCA) insoluble fraction by 98%. However, when the drug is added midway through germination and followed by a pulse labelling period of 1 h, only 65% inhibition of RNA synthesis is observed. This lack of complete inhibition may occur because the drug requires metabolic activation; thus, new rounds of RNA synthesis may have initiated before the drug became fully activated. 4NQO also blocks the de novo expression of beta-glucosidase activity when added early in germination. Additionally, we observe that vegetative cellular slime mold cells are 100 times more resistant than spores to 4NQO-induced damage. Taken together, our results support the observation that RNA synthesis is only required for the emergence stage of germination and that dormant D. discoideum spores may lack efficient excision repair mechanisms.     

Characterization of products formed during the autoxidation of beta-carotene

The anticarcinogenic action of carotenoids such as beta-carotene has been frequently ascribed to their antioxidant properties. However, very little is actually known about the nature of the antioxidant reaction or the products that are formed. beta-Carotene was exposed to either spontaneous autoxidation conditions or to radical-initiated autoxidation conditions. The products were separated by reverse-phase HPLC, and individual peaks were characterized with an on-line diode array detector. Carbonyl products were isolated and characterized by several procedures, including borohydride reduction to the corresponding alcohols, derivatization with O-ethyl-hydroxylamine to the corresponding O-ethyl-oximes of the carbonyls, and analysis by GC-MS. Under the conditions of the experiments, the formation of a homologous series of carbonyl products was demonstrated, including beta-apo-13-carotenone, retinal, beta-apo-14'-carotenal, beta-apo-12'-carotenal, and beta-apo-10'-carotenal. Several very hydrophobic compounds were formed, which have not been previously identified. In addition, the products of NaOCl-treatment of beta-carotene were analyzed, and shown to be significantly different from the autoxidation products. This type of product analysis should be useful in determining the nature of the oxidants reacting with beta-carotene in vivo.     

Reaction products formed after strong acid treatment of alpha-amino-delta-hydroxyvaleric acid

High-voltage paper electrophoresis and analytical and preparative amino acid ion-exchange chromatography were used to investigate the products resulting from strong acid treatment and strong acid treatment and subsequent alkalinization of alpha-amino-delta-hydroxyvaleric acid. After strong acid treatment, alpha-amino-delta-hydroxyvaleric acid and proline, as well as several unidentified components, presumably lactones, were recovered. No definitive identification of the putative unstable lactones was accomplished either by physical-chemical means or by ion-exchange chromatography or high-voltage paper electrophoresis. Nevertheless, after acid treatment and alkalinization, only alpha-amino-delta-hydroxyvaleric acid and proline were recovered, in approximately equal amounts. The formation of such large amounts of proline from the acid treatment and alkalinization of alpha-amino-delta-hydroxyvaleric acid is therefore an important consideration in quantitating the number of gamma-glutamyl phosphate residues present in various proteins.     

Analysis of products formed during bleomycin-mediated DNA degradation

By the use of DNA, copolymers of defined nucleotide composition, and a synthetic dodecanucleotide having putative bleomycin cleavage sites in proximity to the 5'- and 3'-termini, the products formed concomitant with DNA strand scission have been isolated and subjected to structural identification and quantitation via direct comparison with authentic synthetic samples. The products of DNA strand scission by Fe(II)-bleomycin include oligonucleotides having each of the four possible nucleoside 3'-(phosphoro-2'-O-glycolates) at their 3'-termini, as well as the four possible base propenals. At least for 3-(adenin-9'-yl)propenal and 3-(thymin-1'-yl)propenal, the products formed were exclusively of the trans configuration.     

Production of ethylene glycol or glycolic acid from D-xylose in Saccharomyces cerevisiae

Applied Microbiology and Biotechnology - The important platform chemicals ethylene glycol and glycolic acid were produced via the oxidative D-xylose pathway in the yeast Saccharomyces cerevisiae....     

Characterization of sarcosylsarcoursodeoxycholic acid formed during the synthesis of sarcoursodeoxycholic acid

This report describes the isolation of sarcosylsarcosine conjugate of ursodeoxycholic acid (UDCA) formed during the synthesis of sarcoUDCA by the mixed anhydride method. The compound was characterized by its chemical ionization mass spectrum. The diamino acid conjugate was formed only when the free amino acid was used for conjugation. This was confirmed by the isolation of glycylglycoUDCA during the conjugation of UDCA with free glycine but not with glycine ethyl ester hydrochloride. Pure sarcoUDCA was prepared by conjugation of UDCA with sarocisine methyl ester hydrochloride while sarcoUDCA on further reaction with the protected sarcosine derivative gave pure sarcosylsarcoUDCA in 52% yield.     

Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition

The synthesis of novel hybrid hydrogels by stepwise copolymerization of multiarm vinyl sulfone-terminated poly(ethylene glycol) macromers and alpha-omega cysteine oligopeptides via Michael-type additions is described. Cross-linking kinetics, studied by in situ rheometry, can be controlled by pH and the presence of charged amino acid residues in close proximity to the Cys, which modulates the pK(a) of the thiol group. These end-linked networks were characterized by their equilibrium swelling in water, by their viscoelastic properties in the swollen state, and by their soluble fraction. It was demonstrated that structure and properties are very sensitive to the preparation state including stoichiometry and precursor concentration and less sensitive to the pH during cross-linking. For each network the concentration of elastically active chains (nu) was calculated from experimentally determined sol fractions using Miller-Macosko theory and compared to values obtained from swelling and rheometry studies and by calculation from Flory's classical network models. Hydrogels were also prepared with varying macromer structures, and their properties were shown to respond to both macromer functionality and molecular weight.     

Microwave absorption in oligomers of ethylene glycol

The dielectric properties of biologically and pharmaceutically important low-molecular weight ethylene glycols H(-OCH2CH2-)n -OH (n = 1,2,4,6) were investigated to clarify the effect of chain length on the dielectric properties. The measurement of dielectric constant and dielectric loss was carried out over the frequency range 200 MHz to 20 GHz at temperatures of 25 degrees C to 55 degrees C. It is found that in these molecules microwave dielectric losses are significant. The dispersion behaviour of these molecules can be represented by Cole-Cole equation. The dielectric properties of these homologous ethylene glycols are discussed in terms of the effects of chain length and intermolecular hydrogen bonds regarding the molecular conformations. These wide frequency range dielectric data have also been discussed in view of the suitable selection of the oligomer of ethylene glycol for cosmetic preparations and other pharmaceutical applications with the intention of protection of the skin from weak microwave radiations present in the surrounding environment. These systematic microwave dielectric data with frequency and temperature variation are not available and are provided in this paper.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

Effect of ethylene glycol and polyethylene glycol on the acid-unfolded state of trypsinogen

Effect of increasing concentrations of two of the polyols, ethylene glycol (EG) and polyethylene glycol (PEG), was studied by near and far circular dichroism (CD), fluorescence emission spectroscopy, and binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show the transition of acid-unfolded trypsinogen from an unordered state to an intermediate state having ordered secondary structure. Interestingly, near-UV CD spectra show some amounts of stabilizing effect on the tertiary structure of the protein also. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on addition of EG and PEG. Maximum ANS binding occurs in presence of 80% EG and 90% PEG (v/v), suggesting the presence of an intermediate or molten globule-like state at high concentrations of the two polyols.     

Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction.     

Thermal stability of collagen fibers in ethylene glycol

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.     

Developmental toxicity of ethylene glycol monopropyl ether in the rat

In order to determine the potential developmental toxicity of ethylene glycol monopropyl ether (EGPE), pregnant rats were exposed to vapor concentrations of 100, 200, 300, or 400 ppm of the compound for 6 hours per day on days 6-15 of gestation. Maternal effects included a slight reduction in red blood cell count and increased mean corpuscular volume and mean corpuscular hemoglobin at the 200-, 300-, and 400-ppm concentrations. Reticulocyte counts and polychromasia of the red blood cells were increased at all exposure levels, while anisocytosis was increased at 300 and 400 ppm and macrocytosis was increased at 200, 300, and 400 ppm. Hematocrit, hemoglobin concentration, platelet, and total and differential white blood cell counts were comparable to those of the controls. Red urine was seen in the females after the first and second exposures to 200, 300, and 400 ppm of EGPE, but not after subsequent exposures. Absolute and relative spleen weights were increased by 200, 300, and 400 ppm EGPE. Histologic changes were seen in the maternal spleen, liver, and thymus, particularly after exposure to 300 and 400 ppm. Kidneys, bone marrow, and mesenteric lymph nodes were normal. Pregnancy rate, number of corpora lutea, implantation sites and viable fetuses per dam, the incidence of resorptions per litter, and the mean fetal body weights were comparable to those of the controls. Gross external, internal soft tissue, and skeletal examinations of the fetuses revealed that EGPE did not produce teratogenicity or significant embryo/fetotoxicity in the rat at vapor concentrations as high as 400 ppm. Variations in the ossification of certain skeletal elements and the incidence of 14th thoracolumbar rudimentary ribs were increased by exposure to 200, 300, and 400 ppm EGPE.