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PubMed1976

Improved method for measurement of serum levels of phenobarbital, carbamazepine, primidone and diphenylhydantoin by gas-liquid chromatography.

A gas-liquid chromatographic method for the simultaneous determination of phenobarbital, primidone, carbamazepine and diphenylhydantoin in human serum following therapeutic doses has been developed. After extraction with chloroform, the anticonvulsant drugs were methylated with phenyltrimethylammonium hydroxide in dimethylformamide at 85 degrees C for gas-liquid chromatography. Linear temperature programming of a 1% OV-17 column was used to achieve separation and quantitation. The procedure described in the present paper is relatively simple, highly specific and sufficiently sensitive for use in routine clinical assays.

Analysis of Variance↗
PubMed1991

Characteristics of hexosaminidase A in homogenates of white blood cells using methylumbelliferyl-N-acetyl-beta-D-glucosaminide-6-sulphate as substrate.

Using methylumbelliferyl-N-acetyl-beta-D-glucosaminide-6-sulphate as a substrate hexosaminidase A activity was measured in leukocyte homogenates with a one-step method without the need of thermoinactivation. According to the buffer system used--acetate or citrate--the enzyme has pH optima of 3.5 and 4, respectively, and is linear during at least 1 h of incubation. The enzyme is resistant to mechanical inactivation by homogenization or to freezing and thawing and is thermostable between 0 and 37 degrees C but it is rapidly inactivated at 50 degrees C. This thermoinactivation is retarded by acidification of the homogenate. The latency of the enzyme is in agreement with its known lysosomal localization. Like most lysosomal enzymes, hexosaminidase A binds to Concanavalin A. After homogenization of leukocytes and repeated freezing and thawing, about 60% of the enzyme is present in the supernatant. Hexosaminidase A activity was stimulated by taurocholate between 0.05 and 0.1 M and inhibited by dimethylsulfoxide, dimethylformamide, N-acetylglucosamine and N-acetylgalactosamine. No pronounced difference was noted in specific activity of a homogenate of mononuclear cells, granulocytes or total leukocytes.

Binding Sites↗
PubMed1990

Reconstitution of the 9 S estrogen receptor with heat shock protein 90.

As a first step in the investigation of the reconstitution of steroid hormone receptor systems, we studied the reconstitution of 9 S estrogen receptor (ER) from purified vero ER, which is the estradiol binding subunit, and heat shock protein 90 (hsp 90). By using a phosphate buffer containing molybdate, thiocyanate, dimethylformamide, glycerol, etc., vero ER could be converted to 9 S ER with hsp 90, but not with the control protein, ovalbumin. Inactivation of ER during the reconstitution was suppressed partially by hsp 90, but not by ovalbumin. Like native 8 S ER, the reconstituted ER was sedimented at about 8.9 S and 4.6 S on glycerol gradient centrifugation in low and high salt buffers, respectively.

Animals↗
PubMed1992

Determination of diclazuril in animal feed by liquid chromatography.

A method is described for the determination of diclazuril (Janssen Research Compound R64433; trademark Clinacox) in chicken feed at the mg kg(-1) level. Compound R062646, a structure analogous to diclazuril, was used as the interna standard. The drug was extracted from food with acidified methanol. Diclazuril was then isolated by means of solid-phase extraction with a cartridge containing a C18 phase. The eluate was evaporated and the residue redissolved in dimethylformamide. An aliquot was injected onto a reversed-phase high-performance liquid chromatographic column and the drug substance quantified at 280 nm by an ultraviolet detector. Extraction (absolute) recoveries of 85% for both internal standard and diclazuril were obtained. The method is suitable for diclazuril concentrations ranging from 0.1 to 1.5 mg kg(-1). Method validation data are presented.

Animal Feed↗
PubMed1995

Determination of morphine and related alkaloids in crude morphine, poppy straw and opium preparations by micellar electrokinetic capillary chromatography.

A rapid method for the determination of morphine and related alkaloids in crude morphine, poppy straw and opium preparations by micellar electrokinetic capillary chromatography (MEKC) has been developed. Morphine, codeine, thebaine, oripavine, papaverine, narcotine, narceine, cryptopine and salutaridine were separated in less than 10 min using a 70 cm x 50 microm I.D. uncoated fused-silica capillary column with a buffer consisting of 10% dimethylformamide, 90% 0.05 M cetyltrimethylammonium bromide, 0.01 M potassium dihydrogen orthophosphate, 0.01 M sodium tetraborate, pH 8.6. An applied voltage of -25 kV and a temperature of 28 degrees C gave the best separation of the alkaloids. Pholcodine was used as the internal standard. The compounds were detected by UV at 254 nm. The levels of morphine and related alkaloids determined by MEKC were in good agreement with those determined by high-performance liquid-chromatography (HPLC). The coefficients of variation for area calculation (%C.V.) for multiple sample and standard injections by MEKC were slightly greater than for HPLC but were still acceptable (morphine content of poppy straw: %C.V. MEKC 1.7%, %C.V. HPLC 0.3%).

Chromatography↗
PubMed1989

125I-ligand for progesterone receptor: 17 alpha-(6'-iodohex-1'-ynyl)-19-nortestosterone.

Progesterone receptor (PgR) is known to be a significant indicator of hormone dependency of a breast tumor and also an important prognostic factor for recurrence and survival. Relatively low specific activity of tritium-labeled ligands makes it difficult to detect the presence of PgR in very small specimens and in specimens with low PgR content. We therefore undertook the synthesis of a radioiodinated progestin. We have synthesized unlabeled 17 alpha-(omega-haloalk-1'-ynyl)-19-nortestosterones with 4, 6, and 8 carbon side chains; these have high affinity for PgR and clear potential for conversion to a 125I form. Since the six-carbon side chain analogue had the highest relative PgR affinity (38% of the affinity of R5020, using PgR from T47D human breast cancer cells), it was selected for preparation of the 125I form by radiohalogen exchange of a omega-bromoalkynyl precursor with carrier-free Na125I. This new ligand was found by Scatchard analysis to have a Kd of 0.47 nM, as compared with [3H]R5020 at 0.22 nM and [3H]progesterone at 2.0 nM. (For affinity studies, all ligands were diluted in dimethylformamide and added to PgR-containing T47D cytosol to give a final DMF concentration of 4%, in order to prevent R5020 and the test compounds from binding nonspecifically to proteins and glass/plasticware.) The new 125I-ligand showed very little instability when stored in ethanol at approximately 10 degrees C even for several months. Thus, 17 alpha-(6'-[125I]iodohex-1'-ynyl)-19-nortestosterone should prove to be a useful ligand for high sensitivity PgR assays.

Binding, Competitive↗
PubMed1994

Simple continuous and simultaneous determination of tetracycline residues.

A new continuous separation method was developed for the determination of five different tetracyclines (oxytetracycline, tetracycline, chlortetracycline, methacycline and doxycycline). A bioassay using minimum medium (MM) seeded with Bacillus subtilis ATCC 6633 was carried out for the detection, with simple extraction from the agar block of the clear inhibition zone on the MM produced by the mixed tetracyclines. The extract was subjected to continuous identification by high performance liquid chromatography using a mu-Bondapack C18 column. The tetracyclines were separated at ambient temperature using a mobile phase of 0.01 M oxalic acid: acetonitrile: N,N-dimethylformamide (74:18:8, v/v/v) at a flow rate of 1.0 ml min-1. A variable-wavelength detector set at 355 nm and recorder set at 4 mm min-1 were used for the detection. The entire mixture was resolved as five peaks with retention times ranging from 2.75 to 9.65 minutes. This continuous, simple and rapid method of detection, extraction and identification may be useful for routine laboratory testing of residual antimicrobial agents in food.

Bacillus subtilis↗
PubMed1979

Bile acid sulfates. III. Synthesis of 7- and 12-monosulfates of bile acids and their conjugates using a sulfur trioxide-triethylamine complex.

The 7- and 12-monosulfates of chenodeoxycholic acid, deoxycholic acid, and cholic acid were prepared by sulfation of the protected bile acids with sulfur trioxide-triethylamine in pyridine overnight and were isolated by precipitation as the p-toluidinium salt after removing the protecting group(s). The taurine conjugates were obtained by conjugating the bile acid sulfates with taurine in hot dimethylformamide (DMF) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). A new procedure of preparing glycine conjugated bile acid sulfates by direct conjugation of the bile acid sulfate triethylammonium salt with ethyl glycinate in boiling chloroform in the presence of EEDQ is also described. The advantage of these procedures over other procedures are their simplicity and their higher yields (tyically above 90%) The thin layer chromatographic mobilities of these sulfates are presented. The influence of side chain and hydroxyl group configurations on the properties of bile acid sulfates is briefly discussed.

Bile Acids and Salts↗
PubMed1982

Controlled alkaline hydrolysis of steroidal alpha-bromoketones: new conditions and synthesis of 2 alpha-hydroxy-3-ones.

Controlled alkaline hydrolysis of 16 alpha-bromo-17-keto steroids 1, 5 and 7 with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu4NOH) and synthesis of 2 alpha-hydroxy-3-ones 11, 13 and 16 by the controlled hydrolysis of the corresponding 2 alpha-bromo-3-ones 9, 12 and 15 are described. Treatment of the bromoketones 1,5 and 7 with potassium carbonate in aqueous acetone or with n-Bu4NOH in aqueous dimethylformamide (DMF) gave 16 alpha-hydroxy-17-ones 3m 6 and 8 in 85-90% yield, respectively. 2 alpha-Hydroxy-3-ones 11, 13 and 16 were obtained by hydrolysis of the corresponding bromoketones 9, 12 and 15 in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively. Deuterium labeling experiments suggested that equilibration between the 2 alpha-bromoketone 9 and the 2 beta-bromo isomer 10 precedes the formation of the ketol 11 in which the true intermediate might be the 2 beta-isomer 10. However, rearranged androstane derivatives, 3 beta-hydroxy-2-one 18 and 20, were stereoselectively obtained by treatment of the bromoketones 12 and 15 with an excess amount of sodium hydroxide.

Hydrolysis↗
PubMed1990

Synthesis of aminosterols and their derivatives.

Reactions of steroidal epoxides such as 5,6 alpha-epoxy-5 alpha-cholestane (I) and its 3 beta-chloro (II) and 3 beta-acetoxy (III) analogs with urea in dimethylformamide afforded 6 beta-amino-5 alpha-cholestan-5-ol (IV-VI), 6 beta-amino-N-formyl-5 alpha-cholestan-5-ol (VII-IX), and 6 beta-amino-N-amido-5 alpha-cholestan-5-ol (X-XII), along with the 5 alpha-cholestane-5,6 beta-diol (XIII-XV). In addition to these compounds, the 3 beta-acetoxy analog also afforded the N-carboxyl derivative (XVI).

Chemical Phenomena↗
PubMed1991

17 alpha-substituted analogs of estradiol for the development of fluorescent estrogen receptor ligands.

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.

Fluorescence↗
PubMed1986

Effect of a mixed function oxidase inducer and inhibitor on monocrotaline pyrrole pneumotoxicity.

Monocrotaline (MCT) produces vascular injury to the lung, pulmonary hypertension, and right ventricular hypertrophy when injected into rats. It is well established that the pneumotoxicity of MCT depends on its hepatic bioactivation to monocrotaline pyrrole (MCTP) and perhaps other toxic metabolites. To test whether MCTP requires further bioactivation, we synthesized this metabolite chemically, confirmed its structure using fast-atom bombardment-mass spectrometry and nuclear magnetic resonance, and injected it into rats previously treated with an inducer or inhibitor of MFOs. Pretreatment with either phenobarbital or SKF-525A did not alter the pneumotoxic effects of an intravenous injection of MCTP. Rats given the same intravenous dose of either MCT, MCT N-oxide, or MCTP responded with toxicity only to MCTP. MCTP added to rat serum in vitro resulted in a color change (Amax = 477 nm) that developed over several seconds, an observation consistent with degradation of MCTP in serum. To explore the possibility that aqueous degradation products might contribute to its toxicity, the same intravenous dose of MCTP was administered to rats in N,N-dimethylformamide (DMF), serum, or saline. Only MCTP administered in in DMF resulted in toxicity. These results support the contention that MCT requires metabolism to MCTP to produce pneumotoxicity and that exposure to aqueous media renders MCTP incapable of causing lung injury.

Animals↗
PubMed1991

An evaluation of procoagulant activity in the peripheral blood of rats treated with monocrotaline pyrrole.

Monocrotaline pyrrole (MCTP), a putative toxic metabolite of the naturally occurring pyrrolizidine alkaloid, monocrotaline, causes pulmonary vascular thrombi that are associated with vascular remodeling, pulmonary hypertension, and right cardioventricular hypertrophy in rats. The thrombi are composed of platelets and fibrin and occur in the absence of vascular necrosis. Since thrombosis may result from excessive procoagulant activity in the systemic circulation, we evaluated the hemostatic system of rats treated with MCTP to determine if a hypercoagulable state developed in the peripheral blood. Male Sprague-Dawley rats received a single, bolus injection of MCTP (3.5 mg/kg) or an equal volume of the N,N-dimethylformamide (DMF) vehicle in the tail vein. Rats were killed at 1, 3, 5, 8, 11, or 14 days after toxin administration, and several markers of lung injury and hemostasis were evaluated. The protein concentration of cell-free bronchoalveolar lavage fluid (BALF) from MCTP-treated rats was slightly elevated at 1 and 3 days. At 5 days the elevation had become more pronounced, and values increased markedly thereafter. The wet lung-to-body-weight ratio and lactate dehydrogenase activity of cell-free BALF from rats treated with MCTP were mildly increased at 3 days. Increases in these markers progressed at 5 days and values reached a plateau thereafter. MCTP-treated rats had moderately increased total nucleated cell counts in BALF at 5 days, and counts increased markedly thereafter. Right cardioventricular hypertrophy was first detected at 8 days in MCTP-treated rats and became more pronounced with time. The prothrombin time and modified prothrombin time of MCTP-treated rats were consistently greater than those of controls. Although statistically different, these values never exceeded the range of normal values. The activated partial thromboplastin time of MCTP and control rats was variable. Only at Day 14 did MCTP-treated rats have significantly longer activated partial thromboplastin times than those of controls, and these values were within the normal range. Rats treated with MCTP had statistically significant elevations in antithrombin 3 activity at Day 8 and of fibrinogen concentration and antithrombin 3 activity at Day 11 that exceeded the normal range. Platelet numbers of both MCTP- and DMF-treated rats were greater than normal on Day 1 but quickly returned to baseline. The plasminogen values of rats treated with MCTP were lower than controls on Day 5 only. These results suggest that the pulmonary vascular thrombi induced by administration of MCTP to rats are not mediated by an excess in procoagulant activity in the peripheral blood.

Animals↗
PubMed1991

Early indications of monocrotaline pyrrole-induced lung injury in rats.

Monocrotaline pyrrole (MCTP), a putative metabolite of the naturally occurring plant toxin, monocrotaline (MCT), causes lung injury, pulmonary hypertension, and right cardioventricular hypertrophy when administered intravenously to rats. The lesions caused by MCTP administration are similar to those caused by MCT but appear to occur on a slightly accelerated time course. To explore the onset and development of lung lesions, male Sprague-Dawley rats were treated with a single, intravenous injection of MCTP, and several markers of lung injury were evaluated at early times after administration. Rats received 3.5 mg MCTP/kg or an equal volume of the vehicle, N,N-dimethylformamide (DMF), via the tail vein at time 0 and were killed at 4, 12, 24, 48, 72, or 120 hr after treatment. Beginning at 4 hr, MCTP-treated rats had increased wet lung-to-body-weight ratios (LW/BW). The LW/BW remained elevated at 12 hr, returned to baseline at 24 hr, then increased steadily over the next few days. At 24 hr, the protein concentration of cell-free bronchoalveolar lavage fluid (BALF) was slightly elevated. Lactate dehydrogenase activity in cell-free BALF samples was moderately increased 48 hr after MCTP. Changes in these markers were modest initially but became much more pronounced by 120 hr. Total nucleated cell counts in BALF were variable but were moderately elevated at 120 hr. Cytologic examination of the BALF samples revealed small but significant infiltrates of segmented neutrophils at 4 hr and relatively large infiltrates of segmented neutrophils and small lymphocytes at 120 hr post-treatment. Mature neutrophils had degenerate cytomorphologic characteristics at both 4 and 120 hr. These results confirm that pronounced lung injury is delayed for several days after a single, intravenous administration of MCTP, but they also indicate that subtle lung injury can be detected using quantitative markers quite early after MCTP administration.

Animals↗
PubMed1990

Carcinogenic effects of some polycyclic aromatic hydrocarbons on the Japanese medaka and guppy in waterborne exposures.

In this paper, we review studies on the carcinogenic effects of two polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene (BaP) and 7,12-dimethylbenz(a)anthracene (DMBA), on the Japanese medaka (Oryzias latipes) and the guppy (Poecilia reticulata). Exposure media were prepared by adding PAHs to water, with and without dimethylformamide (DMF) as a carrier, and passing this solution through various sized filters. The low exposure medium was a 0.45 micron filtrate without DMF that contained less than 5 micrograms L-1 PAH. The intermediate medium was a DMF-mediated 0.45 micron filtrate which contained 30-50 micrograms L-1 of PAH. The high medium was a DMF-mediated glass-fibre filtrate which contained 150-250 micrograms L-1 of PAH. Young fish specimens (6-10 d old) were given a 6 h exposure once weekly for 2 to 4 wk. Both BaP and DMBA induced hepatic neoplasms in the two species, with the medaka being more sensitive than the guppy, and DMBA being a stronger carcinogen than BaP. Liver neoplasms almost exclusively developed after exposure to BaP and the neoplasms were limited to the high concentration exposure, whereas DMBA caused a substantial number of extrahepatic neoplasms as well as hepatic ones, especially in the medaka. Furthermore, all three concentrations of DMBA induced hepatic neoplasms in the medaka and all but the low concentration induced neoplasms in the guppy. These studies demonstrate the carcinogenic effects of two waterborne PAHs on two small fish species following brief exposures to very low concentrations, and support the contention that environmental PAHs can contribute to the occurrence of cancer in wild fishes.

Animals↗
PubMed1988

Stimulated production of urokinase and plasminogen activator inhibitor-2 by the human promyelocytic leukemia cell line HL-60.

The effects of maturation inducing agents on the production of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) by the human promyelocytic leukemia cell line HL-60 were examined. PA activity, which was calibrated with a urokinase standard, was 3-6 mU/10(6) cells when measured in supernatants from control cells. This activity increased at least two-fold after dimethylformamide (DMF) or retinoic acid (RA) was added to cell cultures, and as much as ten to thirty-fold when cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (PMA), an agent that induces monocytoid differentiation in HL-60 cells. The PA activity produced by control and induced cells had the same molecular weight as urokinase (UK), and was completely inhibited by antibodies to UK. Cells that were induced with PMA but not with RA or DMF also produced an inhibitor to UK that was identified as PAI-2, the plasminogen activator inhibitor that is produced by monocytes. Because of its dual capacity to produce both UK and PAI, the HL-60 cell line represents a useful model for studies of the fibrinolytic mediators that are generated and released by leukemia cells.

Cell Adhesion↗
PubMed1979

Manipulation of myogenesis in vitro: reversible inhibition by DMSO.

A system has been developed for the detailed analysis of the transition from proliferative myoblast to differentiated muscle cell. Dimethylsulfoxide (DMSO) prevents the terminal differentiation of L8 myoblasts in vitro, and its effect is reversible. DMSO (2%) inhibits the fusion of myoblasts to form multinucleate myotubes, the normal increases in activity of creatine phosphokinase (CPK) and acetylcholinesterase, and the synthesis of alpha-actin and acetylcholine receptor protein. Upon removal of DMSO from the medium, a lag precedes the onset of differentiation. The potential to inhibit muscle differentiation reversibly is not specific to DMSO, but is shared by a number of compounds, including dimethylformamide, hexamethylbisacetamide and butyric acid, all potent inducers of gene expression in Friend erythroleukemia cells. L8 cells routinely cease DNA synthesis and initiate fusion and muscle protein synthesis once they are confluent. In the presence of DMSO, however, nearly all cells continue DNA synthesis, even several days after reaching confluence. Protein synthetic patterns of DMSO-inhibited cells are almost indistinguishable from those of untreated myoblasts and distinct from differentiated myotubes. It appears that cells exposed to DMSO are locked indefinitely in a proliferative myoblast stage of development and are unable to enter the Go phase of the cell cycle necessary for initiation of differentiation. DMSO coordinately inhibits all the differentiative parameters measured. In contrast, cytochalasin B uncouples normally linked differentiative events so that fusion is inhibited while muscle-specific protein synthesis proceeds. DMSO has similar effects on both cytochalasin B-treated and fusing control cultures, suggesting that its primary effect is exerted not at the level of fusion but earlier in the differentiative time-table. Once fusion and the synthesis of muscle-specific proteins are well under way, the addition of DMSO is ineffective and differentiation continues in its presence. The potential to manipulate muscle gene expression in vitro makes this system particularly useful for the detailed analysis of the processes involved in the transition to the differentiated state and for determining the linkage of developmental events.

Actins↗
PubMed1993

Borate ion-assisted stabilization of beta-galactosidase from Aspergillus oryzae by polyhydroxy compounds in water-miscible organic solvents.

The stability of beta-galactosidase from Aspergillus oryzae in water-miscible organic solvents in different buffers at various pH values ranging from 4.6 to 8.0 was studied. The stability of the enzyme in all six organic solvents studied was dependent on pH and on the type of buffer ions present. At a given pH, destabilization by organic solvents was highest in sodium borate buffer. The destabilization of beta-galactosidase by these solvents could be reversed by addition of sugars or polyhydroxy compounds exclusively in sodium borate, suggesting a role of borate ions in stabilization. A similar effect of addition of mannitol was observed on deactivation of beta-galactosidase by N, N-dimethylformamide (DMF). Exclusively in sodium borate, at pH 8.0, the addition of mannitol (0.02 M) not only prevented the deactivation by DMF (8%, v/v) but increased the enzyme activity to the level at its optimum pH. Since beta-galactosidase from Aspergillus oryzae is a glycoprotein, complexation of the borate ions to the carbohydrate part may result in change in protein conformation, which, without leading to denaturation or inactivation of the enzyme, may facilitate interaction of the organic solvents with the enzyme leading to its denaturation. Such a denaturation is probably prevented by addition of polyhydroxy compounds, which appear to compete favorably with the carbohydrate moiety of the protein in complexing with borate ions. This should result in the enzyme regaining its native conformation.

Aspergillus oryzae↗