Removal of triethylamine from synthetic wastewater by acclimated mixed bacteria cultures

This aim of this study was to remove triethylamine by a biological method, as well as to understand the ability of mixed bacteria cultures to treat a triethylamine compound from synthetic wastewater. The mixed bacteria cultures could not remove triethylamine, whether the activated sludge came from an acrylonitrile–butadiene–styrene resin manufactured wastewater treatment system or a waterborne polyurethane resin manufactured wastewater treatment system. When the mixed bacteria cultures were acclimated to triethylamine, they could utilize 650 mg l⁻¹ triethylamine for growth. When the initial triethylamine concentration was below 200 mg l⁻¹, the triethylamine removal efficiency could reach 100%. The triethylamine removal rate of the acclimated GMIX sludge was faster than the acclimated EMIX sludge.     

A new approach to the synthesis of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotide analogues.

A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology.     

Sequence analysis of end-labeled DNA fragments by solvolysis in aqueous solutions of different amines.

Cleavage of 3'-end-labeled DNA in hot aqueous solutions of different amines is comparatively examined for overall rate of DNA scission as well as for potential differences in the preference of the various amines for cleavage at the different bases. Under comparable conditions (0.5 M amine, 0.3 M NaCl, 90 degrees C), piperidine, diethylamine, morpholine, and ethylenediamine produce the same set of labeled fragments, at approximately equal overall cleavage rates. The same set of fragments is also obtained with diisopropylamine, triethylamine, and 1,4-diazabicyclo[2.2.2]octane, but at markedly lower overall cleavage rates. Solvolysis in aqueous piperidine or aqueous diethylamine leads to DNA scission predominantly at A sites, followed by G and C sites, and least frequently at T sites. In contrast, morpholine, ethylenediamine, diisopropylamine, triethylamine, and diazabicyclo[2.2.2]octane cleave the DNA predominantly at G sites. Therefore, use of one of the latter amines allows clear distinction of G bands and C bands, which could not be distinguished by the criterion of band intensity in the original one-lane sequencing method based on cleavage in hot aqueous piperidine (B. Ambrose and R. Pless (1985) Biochemistry 24, 6194-6200). The effect of varying the salt concentration on the cleavage distribution obtained with various amines is also examined, and a rationale is given for the influence of salt concentration and amine basicity on the relative rate of cleavage at G sites.     

High-performance liquid chromatographic determination of pipotiazine in human plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of pipotiazine in human plasma and urine. After selective extraction, pipotiazine and the internal standard (7-methoxypipotiazine) are chromatographed on a column packed with Spherosil XOA 600 (5 μm) using a 7:3 (v/v) mixture of diisopropyl ether—isooctane (1:1, v/v) + 0.2% triethylamine and diisopropyl ether—methanol (1:1, v/v) + 0.2% triethylamine + 2.6% water. The eluted compounds are measured by fluorescence detection. The sensitivity of the method was established at 0.25 ng/ml pipotiazine in plasma and 2 ng/ml pipotiazine in urine (C.V. < 5%). The method has been successfully applied to a pharmacokinetic study following a single oral administration of 10 mg of pipotiazine.     

Fmoc-amino acid chlorides in solid phase synthesis of opioid peptides.

Fmoc-amino acid chlorides were employed in the solid phase synthesis of the opioid peptides [Leu]enkephalin, [Leu]enkephalin amide, and dermorphin. The conventional polystyrene-based Merrifield resin or Wang's resin served as solid support. A binary salt of either triethylamine or diisopropylethylamine in the presence of 1-hydroxybenzotriazole or pivalic acid was used for acylation. The coupling rates were quite fast, being comparatively faster when 1-hydroxybenzotriazole was used along with triethylamine or diisopropylethylamine. The peptides obtained in good yields showed, after purification, biological and spectral properties identical with those of the natural peptides.     

Phosphono-platelet activating factor. II. Synthesis of 2-acetamido-2-deoxy-1-O-octadecylglycerol-3-(2-trimethylammoniumethyl) and (2-aminoethyl)phosphonates

The chemical synthesis of the amide analogs of 1-O-alkyl-2-O-glyceryl-3-O-phosphoryl choline as its phosphono analog (phosphono-AGEPC) and 1-O-alkyl-2-O-acetyl-glyceryl-3-O-phosphoryl ethanolamine as its phosphono analog (phosphono-AGEPE) is reported.The intermediate acetamides for the subsequent phosphonylations were obtained (i) by classical organic reactions and (ii) by the method of Chandrakumar and Hadju (Tetrahedron Lett., 23 (1982) 1043–1046). Phosphonylation for the choline analog was accomplished with 2-bromoethyl phosphonic acid monochloride in anhydrous and ethanol-free chloroform in the presence of triethylamine. This was followed by reaction with anhydrous trimethylamine in dimethylformamide in a sealed tube at 50–55°C for 3 days.Phosphonylation for the ethanolamine analog was accomplished with 2-pinthalimidoethyl-phosphonic acid monochloride in anhydrous and ethanol-free chloroform in the presence of anhydrous triethylamine, followed by hydrazinolysis in 90% ethanol under reflux for 4 h. The products were identified by elemental analysis, thin-layer chromatography (TLC) and IR spectroscopy.     

An HPLC procedure for separating polyphosphoinositides on hydroxylapatite

We describe a method for separation of several phosphoinositides by high performance liquid chromatography (HPLC) for the purposes of identification, analysis, and possible purification of cell phospholipid extracts. The phosphoinositides were separated on an hydroxylapatite column using a solvent system consisting of tetrahydrofuran-ethanol-water with a gradient of triethylamine phosphate ranging from 1 to 100 mM. Increasing triethylamine phosphate concentrations over a series of isocratic steps resulted in the resolution of [3H]phosphoinositide standards (phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate) and their lyso-derivatives. The eluted peaks were collected and quantitated using scintillation counting, and the samples of the eluates were subjected to further analysis by thin-layer chromatography to verify their integrity and identity.     

Radiochemical methods for the assay of phosphorylase in the direction of glycogenolysis.

Two radiochemical procedures were explored for the determination of phosphorylase activity in the glycogenolytic direction. In the "32P assay method' the formation of labelled glucose 1-phosphate from glycogen and [32P]Pi is measured by the radio-activity that remains soluble after the precipitation of phosphomolybdate with triethylamine. In the "14C assay method' the formation of labelled glucose 1-phosphate from peripherally 14C-labelled glycogen and P1 is determined from the radioactivity that remains soluble after the precipitation of glycogen with ethanol. The 14C assay method requires more preparative work but less circumspection than does the 32P assay method. Both radiochemical methods can be applied where the classical spectrophotometric assay fails. They have the same accuracy and reproducibility, and allow more samples to be handled in parallel. They are not intended for use with crude tissue extracts.     

Reaction of phenylglyoxal with arginine. The effect of buffers and pH.

Phenylglyoxal reacts much more rapidly with N²-acetylarginine than with either N²-acetyllysine or N-acetylcysteine. The rate of the reaction of phenylglyoxal with either N-acetylarginine or arginine increases with increasing pH from 7.5 to 11.5. The model reaction with arginine is much faster in bicarbonate, diethylamine, or triethylamine buffer than in N-ethylmorpholine, borate, phosphate, or Tris buffer. This activation by various buffers should be taken into consideration when glyoxal derivatives are used to modify arginyl residues.     

Possible role of hydrogen cyanide in chemical evolution. The oligomerization and condensation of hydrogen cyanide

The rate of cyanide oligomerization is independent of the presence of added nucleophiles such as azide, monomethylamine or triethylamine and is dependent only on the pH of the reaction mixture. The products formed, with the exception of urea, are also independent of the nucleophiles used to initiate reaction. In the presence of monomethylamine, monomethylurea is the main neutral product instead of urea, suggesting the intermediacy of cyanate. Evidence is presented which suggests that cyanogen may be the precurser to both cyanate and oxalic acid in the cyanide Oligomerization.     

The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation.

Reaction of bovine erythrocyte superoxide dismutase with H2O2 was accompanied by a luminescence whose intensity was a function of the concentration of H2O2 and whose duration was coincident with the inactivation of the enzyme by this reagent. Oxygen, which protected against inactivation, also diminished the luminescence. Several other compounds which prevented the inactivation by H2O2 also modified the luminescence. Thus urate, formate, and triethylamine inhibited luminescence whereas imidazole and xanthine augmented it. These seemingly contrary effects can be explained by assuming that the compounds which protected the enzyme were peroxidized in competition with the sensitive group on the enzyme. The luminescence arises because that group on the enzyme was oxidized to a product in an electronically excited state, which could return to the ground state by emitting light. Imidazole and xanthine gave electronically excited products whose quantum efficiency was greater than that of the group on the enzyme, whereas urate, formate, and triethylamine gave products with much lower luminescent efficiencies. This superoxide dismutase could catalyze the peroxidation of a wide range of compounds, including ferrocytochrome c, luminol, diphenylisobenzofuran, dianisidine, and linoleic acid. In control experiments, boiled enzyme was inactive. This peroxidative activity can lead to unexpected effects when superoxide dismutase is added to H2O2-producing systems, as a probe for the involvement of O2-. Several examples from the literature are cited to illustrate the misinterpretations which this previously unrecognized peroxidative activity can generate.     

甲氨基阿维菌素苯甲酸盐微乳剂热贮稳定性研究

采用高效液相色谱法分析甲维盐微乳剂,以甲醇＋0．2％三乙胺溶液＋乙腈（V：V：V=30：30：40）为流动相,采用XterraRP184．6150mm色谱柱,检测波长245nm,测得54±2℃条件下14d分解率为3．42％。室内毒力测定热贮前后1％甲维盐微乳剂Lc50分别为0．499mg／L和0．524mg／L,热贮分解率为4．78％,与高效液相色谱测定结果基本一致。     

Immobilized enzyme cellulose hollow fibers: I. Immobilization of heparinase

The immobilization of heparinase to tresyl-chloride-activated cellulose hollow fibers for the removal of heparin from the bloodstream was examined. Whole blood can be circulated through cellulose hollow fibers without hemolysis and the tresyl chloride chemistry provides a strong linkage which limits the release of the enzyme from the support. The tresylation and immobilization methods were modified and optimized to improve the heparinase activity retained by cellulose. Pretreatment of the hollow fibers with 0.05/V sodium hydroxide increased the degree of tresylation and the immobilization yield by a factor of five. The use of triethylamine as the organic base in the tresyl chloride activation resulted in threefold greater activity retention by the support than when pyridine was used. Together, sodium hydroxide pretreatment and triethylamine enhanced the activity retained by cellulose to 26.2 +/- 7.0% of that bound to the support. The activity retention was also a function of the technique used for immobilization. The best results were achieved when the enzyme was applied to the activated fibers once every 12 to 24 h for a total of four times. The active enzyme loading on the fibers was 0.3 mg heparin degraded/h cm(2) when 4.5 mug protein/cm(2) was bound to the fibers.     

An efficient method for conjugation of a lipopolysaccharide from Salmonella enterica sv. Minnesota with probes bearing hydrazine or amino functional groups

A conjugation method for coupling probes bearing hydrazine or primary amino groups to a lipopolysaccharide (LPS) is described. LPS is modified through the hydroxyl groups present in its O-antigen moiety by activation with cyanogen bromide in aqueous acetone using triethylamine to enhance the electrophilicity of CNBr. The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety, which in blood exerts pleiotropic effects on many tissues and organs, resulting in multiple-organ damage, circulatory collapse, and death. Conjugation of smooth-form LPS from Salmonella enterica sv. Minnesota to dansyl hydrazine yielded a labeling ratio of 330 nmol dansyl/mg LPS, with nearly no loss of the original endotoxic activity. In the case of horseradish peroxidase, in which a spacer was introduced, the ratio was 28 nmol HRP/mg of LPS, preserving 65% of the original endotoxic activity. This work shows that under these conditions of CNBr activation, the labeling process has practically no effect on the endotoxic behavior of LPS. The method can be used effectively for the conjugation of LPS to probes bearing primary amino, hydrazine, or hydrazide functional groups.     

A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues.

A simple and rapid method for a highly sensitive radioimmunoassay of cyclic AMP and GMP is described. The method is based on the observation that the affinity of the cyclic nucleotide antibodies for the 2′-0-succinyl or acetyl derivatives is considerably greater than that for the nonacylated cyclic nucleotides. With the present method, 3–10 fmoles of cyclic AMP and cyclic GMP can be assayed using commercially available antisera against cyclic AMP or cyclic GMP. A reproducible conversion of the cyclic nucleotides in aqueous samples to the 2′-0-acylated derivative is brought about by a simple one-step addition of premixed reagents containing either succinic anhydride or acetic anhydride and triethylamine. The time required for succinylation or acetylation of 100 samples is less than 5 min using this technique. Tissue extracts after purification through anion exchange resins do not interfere with the acylation. After acylation, labeled antigen and antiserum are added and incubated at 4°C for 15 hr. The bound antingen is separated from the unbound antigen by a simple ethanol precipitation using bovine serum albumin to ensure complete precipitation. The specificity of the method has been validated by the following criteria: (1) treatment of the samples with purified phosphodiesterase results in complete loss of cyclic nucleotide immunoreactivity, (2) cyclic nucleotide content was a linear function of the tissue weight, and (3) in one instance using bovine rod outer segments, identical values of cyclic GMP were obtained with the present method and a purely physical method (high pressure liquid chromatography).     

A simple conductimetric method as an alternative to the colorimetric p-nitrobenzylpyridine test for the measurement of the reactivity of potentially mutagenic alkylating compounds

A simple method for the measurement of the kinetics of reaction of potentially mutagenic alkyl halides with amines, based on the direct conductimetric monitoring of the quaternary ammonium salt produced in these reactions, is proposed and applied to the alkylation of p-nitrobenzylpyridine (NBP) and triethylamine (TEA) in different solvents. With respect to the classical colorimetric NBP-test, this method has the advantage that the rates can be measured continuously over the entire course of the reactions and the kinetic order and constants can be easily obtained. It is also shown that the previously proposed, NBP modified test', using simultaneously NBP and TEA, gives actually the sum of the rate constants for the reactions of the alkylating reagent with the two amines.     

Application of high performance liquid chromatography to the preparation of encephalitogenic myelin basic protein

Two preparations of myelin basic protein (MBP) were derived from an acid excretion of chloroform-methanol defatted bovine spinal cord. The first was purified by ion-exchange chromatography using guanidine-HCl; the second, by high performance liquid chromatography (HPLC) using a triethylamine eluant. Both methods of preparation yield MBP which is identical on acid-urea polyacrylamide gel electrophoresis and which has identical encephalitogenic potency. Because of the greater time-efficiency of the HPLC system with no deleterious side effects due to buffer contamination, this latter method can be recommended for MBP purification.     

Novel epoxide formation in the reaction of 2-bromo-3-methyl-1,4-naphthoquinone with 1,3-propanedithiol

A novel epoxide 2 was formed as the major product in the reaction of 2-bromo-3-methyl-1,4-naphthoquinone with 1,3-propanedithiol in the presence of triethylamine in 92% yield. Molecular oxygen is suggested to be the source of the added oxygen in 2, an oxidation product of its precursor 3. A strong base such as triethylamine is required to abstract the methyl hydrogen of 1,4-naphthoquinones, leading to the formation of 3 as well as 2.     

Determination of fifteen nucleotides in cultured human mononuclear blood and umbilical vein endothelial cells by solvent generated ion-pair chromatography

The paper describes the development of a method for the determination of 15 nucleotides in cultured mononuclear blood and umbilical vein endothelial cell lysates by solvent generated ion-pair chromatography. The phase system is generated via a mobile phase of 100 mM phosphoric acid adjusted to pH 6.2 with triethylamine. Nucleotides are eluted by applying a linear magnesium ion gradient. The method is robust, highly reproducible and easily adaptable to other cell lysates and allows the separation and quantitation of the nucleotides with detection limits in the range from 17 (ADP) to 126 (CDP) pmol in 20-microl aliquots.     

Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis.

A method is described for the isolation of peptide fragments from proteins separated by polyacrylamide gel electrophoresis. After completion of the electrophoresis step, gels are stained with Ponceau S or Coomassie Blue. Gel portions containing protein stained with Ponceau S are excised and transferred to borosilicate glass digestion tubes containing 0.9 ml of 1 mM NaOH or 5 mM Na2HPO4. After complete dissociation of the dye from the protein, 0.1 ml of 20% formic acid is added and the protein is hydrolyzed in situ at 112 degrees C for four hours. Subsequently the acid solution is made 10% in acetonitrile and chromatographed as such on a C18 (C4) reversed-phase column using an appropriate large-volume sample loading syringe and injection loop. Proteins stained with Coomassie Blue can be hydrolyzed in situ after complete removal of the dye with an aqueous solution containing 40% acetone, 10% triethylamine and 5% acetic acid. The gel slices are next washed with HPLC-grade water and protein is hydrolyzed in 2% formic acid under standard conditions. Gel-related contaminants do not interfere with the peptide separation under the proper conditions of HPLC analysis.