Isolation and Restriction Mapping of a Pseudomonas Plasmid

The effects of the injection of a large amount of N¹ -methylnicotinamide (MNA) (500 mg per kg body weight) on the ratio of N¹ -methyl-4-pyridone-3-carboxamide (4-py) to N¹ -methyl-2-pyridone-5- carboxamide (2-py) excretion, and the activities of 2-py and 4-py forming MNA oxidases were investigated in rats. The injected MN A was excreted very rapidly into the urine; 46 % of the dose was excreted from 0~3hr post-injection, 15% from 3~6hr, 6% from 6~9hr and 1.5% from 9~ 12hr. The ratio of 4-py to 2-py also decreased rapidly; the ratio being about 0.6, 0.4, 0.4 and 0.6 from 0~3hr, 3~6hr, 6~9hr and 9~ 12hr post-injection, respectively. This ratio then recovered rapidly; being about 2, 5.5, 8.5 and 9.7 from 12~24 hr, 24 ~48 hr, 48~72 hr and 72 ~96 hr post-injection, respectively. The normal range of 4-py to 2-py excretion ratio is 8~14. So, this ratio returned to a normal level by day 3 post-injection. The rats were killed 5 hr after the MNA injection. At this time (the lowest ratio was observed around this time), the activities of 2-py and 4-py forming MNA oxidases in the injected group were 59 % and 11 % of the normal levels, respectively. Therefore, it was found that the decreased ratio of 4-py to 2-py excretion with the MNA injection was mainly due to the higher inhibition of the 4-py forming MNA oxidase than of the 2-py forming MNA oxidase by the MNA injection.     

Excretion of estrone,estradiol, and progesterone in the urine and feces of the female cotton-top tamarin (Saguinus oedipus oedipus)

The excretion of three gonadal steroids was studied in the urine and feces of female cotton-top tamarins (Saguinus oedipus oedipus). Each steroid, ¹⁴C-estrone, ¹⁴C-estradiol, and ¹⁴C-progesterone, was injected into a separate female cotton-top tamarin. Urine and feces were collected at 8 hr intervals for 5 days on the three tamarins. Samples were analyzed to determine the proportion of free and conjugated steroids. Steroid excretion patterns were determined by sequential ether extraction, enzyme hydrolysis, and chromatography. Labeled estrone was excreted in a slow and continuous manner into the urine (57%) and feces (43%) with 90% of the steroid conjugated. The nonconjugated form had an elution profile identical to ³H estrone, but the conjugated portion was not completely hydrolyzed by enzyme. Labeled estradiol was excreted primarily in the urine (87%) and was released rapidly. Over 90% of the injected ¹⁴C-estradiol was excreted in urine as a conjugate, of which 41% was converted to an estrone conjugate and the remaining 59% was excreted as a polar estradiol conjugate. Labeled progesterone was excreted primarily in the feces (95%), 61% of which was free steroid. Four to six individual peaks of radioactivity were found when using celite chromatography and high performance liquid chromatography (HPLC), indicating that progesterone is metabolized into several urinary and fecal metabolites. One of these peaks matched ³H-progesterone and others may be pregnanediols, pregnanetriols, and 17-hydroxyprogesterone. These steroidal excretion patterns help explain the atypical hormonal patterns seen during the tamarin ovarian cycle.     

The metabolism of arylazoisoxazolones by rats and dogs

1. When rats were given a single oral dose of the lipid-soluble fungicide 4-(2-chlorophenylhydrazono)-3-methyl[4-(14)C]isoxazol-5-one ([(14)C]drazoxolon), about 75% of the label was excreted in the urine and 13% in the faeces in 96hr. An additional 7% of the radioactivity was recovered as (14)CO(2) in 48hr. 2. About 8% of the label was excreted by rats in the bile in 0-24hr. and an additional 6% was excreted by the same route in 24-48hr. 3. When dogs were given a single oral dose of [(14)C]drazoxolon about 35% of the label was excreted in the urine and a similar amount was excreted in the faeces in 96hr. 4. The major metabolites in the urine of the rat and the dog were identified as 2-(2-chloro-4-hydroxyphenylhydrazono)acetoacetic acid (dog, 14%), the corresponding ether glucosiduronic acid (dog, 12%; rat, 13%) and ester sulphate (rat, 65%). 5. When rats were given a single oral dose of 3-methyl-4-([U-(14)C]phenylhydrazono)isoxazol-5-one about 75% of the label was excreted in the urine and 15% in the faeces in 96hr. The major metabolite in the urine was identified as the ester sulphate conjugate of 2-(4-hydroxyphenylhydrazono)-acetoacetic acid. 6. Reduction of the azo link was of minor quantitative significance. 7. These results are discussed in their relation to species differences in the toxicity of these compounds.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

A STUDY OF LIGHT INTENSITY,PERIODICITY, AND WAVELENGTH ON ZOOSPORE PRODUCTION BY PROTOSIPHON BOTRYOIDES KLEBS12

When mature Protosiphon cells were placed in darkness, zoospore production was more extensive and was completed in a shorter time at a temperature of 27 C than at 22 or 15 C. Cool-white fluorescent (Sylvania) light inhibited the process measurably at a radiation intensity of 0.6±10³ ergsjcm²-sec; inhibition was 96% complete at 14±10³ ergs/cm²-sec. For mature cells previously grown under repeated 12-12 hr light-dark cycles, a dark period of approximately 2 hr at 22 C allowed cell division to proceed to a stage such that reillumination did not inhibit continued development of zoospores. Monochromatic light from 402 to approximately -494 nm, as compared to darkness, inhibited zoospore formation; maximal inhibition was at 432-461 nm. In contrast, monochromatic light from 522 to 726 nm stimulated zoospore formation relative to darkness. Synchronous zoospore production was obtained using the following regimes: (A) 12 hr cool-white alternated with 12 hr yellow, (B) 12 hr cool-white alternated with 12 hr blue. Under regime A synchronous zoospore release (following synchronous production) occurred near the end of the yellow irradiation period, while under regime B it occurred near the end of the cool-white irradiation period. The significance of this in terms of photoprocesses and possible photoreceptors is discussed.     

Metabolism of Isoxathion,O, O-Diethyl O-(5-Phenyl-3-isoxazolyl) phosphorothioate in the Rats

Excretion, distribution and metabolism of the insecticide, Isoxathion, administered orally in male Wistar-strain rats, were investigated with a carbon-14 labeled chemical. During 96 hr, approximately 85% and 14% of the total radioactivity were excreted in the urine and feces. Distribution of isoxathion after oral administration in the rats was investigated by means of whole-body autoradiographic technique and measurement of radioactivity in the tissues. At least eleven radioactive metabolites were detected, four of which were structurally determined. They were 3-hydroxy-5-phenylisoxazole, 3-(β-d-glucopyranuronosyloxy)-5-phenylisoxazole, 5-phenyl-3-isoxazolyl sulfate and hippuric acid.     

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-apomorphine a 10 μm Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min⁻¹ the total run time is ca. 15 min. The detection limits are 0.3 and 0.6 ng ml⁻¹ for R- and S- apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml⁻¹ for plasma samples, and <4% in the concentration range of 40-400 ng ml⁻¹ for urine samples. For the assay of apomorphine, apocodeine and isoapocodeine, a 5 μm C₁₈ column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a flow-rate of 0.8 ml min⁻¹ the total run time is ca. 14 min. The detection limits of this assay are 1.0 ng ml⁻¹ for apomorphine and 2.5 ng ml⁻¹ for both apocodeine and isoapocodeine (signal-to-noise ratio 3). The inter-assay variations are 5% in the concentration range of 5-40 ng ml⁻¹ for plasma samples and 7% in the concentration range of 50-500 ng ml⁻¹ for urine samples. The glucuronic acid and sulfate conjugates of the various compounds are hydrolysed by incubation of the samples with β-glucuronidase and sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of apomorphine, apocodeine and isoapocodeine occurred during the incubation. A pharmacokinetic study of apomorphine, following the intravenous infusion of 30 μg kg⁻¹ for 15 min in a patient with Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent drug and the appearance of apomorphine plus metabolites in urine could be determined.     

Analysis of the metabolites of the sodium salt of 6-hydroxy-5-(phenylazo)-2-naphthalenesulfonic acid in Sprague–Dawley rat urine

The sodium salt of 6-hydroxy-5-(phenylazo)-2-naphthalenesulfonic acid (SS-AN), which is a subsidiary color present in Food Yellow No. 5 [Sunset Yellow FCF, disodium salt of 6-hydroxy-5-(4-sulfophenylazo)-2-naphthalenesulfonic acid], was orally administered to Sprague–Dawley rats. Metabolite A, metabolite B, and unaltered SS-AN were detected as colored metabolites in the rat urine. Analysis of the chemical structures showed that metabolite A (major peak) was 6-hydroxy-5-(4-sulfooxyphenylazo)-2-naphthalenesulfonic acid, the sulfuric acid conjugate of SS-AN, and metabolite B (minor peak) was 6-hydroxy-5-(4-hydroxyphenylazo)-2-naphthalenesulfonic acid (SS-PAP), which is a derivative of metabolite A without the sulfuric acid. The colorless metabolites p-aminophenol, o-aminophenol, and aniline present in the urine were analyzed by liquid chromatography–mass spectrometry. The orally administered SS-AN had been metabolized to the colorless metabolites (p-aminophenol 45.3%, o-aminophenol 9.4%, aniline 0.4%) in the 24-h urine samples. Analysis of the colored metabolites by high-performance liquid chromatography with detection at 482 nm indicated the presence of metabolite A (0.29%), SS-PAP (0.01%), and SS-AN (0.02%) were detected in the 24-h urine samples. Approximately 56% of SS-AN was excreted into the urine and the rest is probably excreted into feces.     

In vitro micropropagation of Acacia nilotica subsp. indica Brenan via cotyledonary nodes

Cotyledonary node explants of Acacia nilotica subspecies indica Brenan, differentiated multiple shoots on Gamborg et al.' s medium (B₅, Gamborg et al. 1968) supplemented with cytokinins like N⁶-benzyladenine, 6-(, -Dimethylallylamino)-purine, kinetin or zeatin. Of the four, BA supported maximum multiple shoot differentiation; the highest average number of shoots (6.3) per expiant was in 1.5 mg/l. The number of shoots was further enhanced by (i) using nodal explants of in vitro regenerated shoots as microcuttings, and (ii) repeated subculture of the original expiants (stumps) on the same medium after excising the shoots. Thus, over seven hundred shoots could be obtained from a single cotyledonary node explant. Individual shoots, when transferred to 2 mg/l indole-3-acetic acid augmented medium organised healthy roots in 100% cultures. Such test tube grown plantlets have been successfully transferred to soil, where they grow well up to eight weeks.Abbreviations 2iP 6-(, -Dimethylallylamino)-purine - AC activated charcoal - B₅ Gamborg et al. 's medium with 0.8% agar + 3 % sucrose - BA,N⁶ benzyladenine - CW coconut water - FAA formalin-aceticacid-alcohol - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn kinetin - NAA -naphthaleneacetic acid - NOA -naphthoxyacetic acid     

THE EFFECT OF ESTRADIOL ON THE CELL KINETICS IN THE UTERINE AND CERVICAL EPITHELIUM OF NEONATAL MICE

The effect of estradiol-17β on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle, T_c, from 17-9 hr to 15-7 hr, and the duration of S phase, T_s, from 6–7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, T_c is shortened to 7-4 hr and T_s to 4–5 hr. The shortening of T_c at 12 hr is mainly due to an effect on T_G1, which is shortened from 8–55 hr in untreated animals to 1–8 hr in estradiol treated animals. The T_c of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the T_c was now increased to 47 hr and further to 61 -2 hr following 12 hr treatment with the hormone. T_s increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in T_G1, which is lengthened from 10–95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

Metabolism of Cyanox®, O,O-Dimethyl O-(4-Cyanophenyl) Phosphorothioate in the Rats

Orally administered Cyanox, O, O-dimethyl O-(4-cyanophenyl) phosphorothioate, labelled with carbon-14 at p-cyano group was easily absorbed from the gastrointestinal tract of male Wistar rats, and distributed into tissues. Elimination of the radioactivity was rapid and essentially complete; namely during 96 hr approximately 90.7% and 10% of total radioactivity were excreted respectively into urine and feces. Carbon-14 in expiration was negligible. No detectable amount of Cyanox was found in the urine and 0.01% of the administered compound was present in the feces. Degradation products in the urine were identified as demethylcyanox, demethylcyanoxon, p-cyanophenol and p-cyanophenylsulfate.     

Cell Size,Macromolecular Composition,and O2 Consumption During Agitated Cultivation of Naegleria gruberi*

SYNOPSIS. Cell size, macromolecular composition, carbohydrate utilization patterns, and O₂ concentrations were measured throughout the growth stages of Naegleria gruberi in agitated cultures in a complex medium. Biphasic logarithmic growth occurred during the initial 83 hr of growth and the mean generation time was 7.0 hr and 19 hr during initial and secondary log growth stages, respectively. The maximum yield was 5 × 10* amebaeJml. The pH rose rapidly (1 pH unit) during the secondary log growth phase (52-83 hr) and continued into the stationary growth phase (83-120 hr). Dry weight, total protein, carbohydrate, and RNA per ameba increased just before the secondary log growth phase. RNA increased 31% to 35% per ameba at the end of each phase of log growth. DNA increased ～ 2-fold throughout the different growth phases. Average cell size increased 90% during biphasic log growth then decreased during stationary phase. O₂ tension decreased from 100% to 18% of saturation during the biphasic growth phase, then increased during stationary growth to near 100% saturation. Glucose and total carbohydrate assays showed little utilization of those substrates throughout the growth stages. Naegleria gruberi presumably has a predominantly aerobic metabolism, also its metabolism may change during the different growth phases.     

Determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine using high-performance liquid chromatography with fluorescence detection

A method for the determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine is described. Plasma and urine samples were extracted using 3M Empore extraction disk cartridges in the C₁₈ and MPC (mixed-phase cation-exchange) formats, respectively. The extract was analyzed using HPLC with fluorescence detection (ex 248 nm, em 300 nm), and included a column switching procedure to reduce run-time. The assay was linear in the concentration range 5 to 1000 ng/ml when 1-ml aliquots of plasma and urine were extracted. Recoveries of L-756 423 were greater than 84% over the calibration curve range using the described sample preparation procedures. Intra-day precision and accuracy for this assay was less than 9% RSD and within 7%, respectively. Inter-day variabilities for the plasma (n=17) and urine (n=10) were less than 5% and 3% for low (15 ng/ml) and high (750 ng/ml) quality control samples. Bovine serum albumin (0.5%) was used as an additive to urine to prevent precipitation of L-756 423 during the storage of clinical samples. The assay was used in support of human clinical trials.     

High-throughput, semi-automated determination of a cyclooxygenase II inhibitor in human plasma and urine using solid-phase extraction in the 96-well format and high-performance liquid chromatography with post-column photochemical derivatization-fluorescence detection

Compound I, 5-chloro-3-(4-methanesulfonylphenyl)-6′-methyl-[2,3′]bipyridinyl, has been found to be a specific inhibitor of the enzyme cyclooxygenase II (COX II). The anti-inflammatory properties of this compound are currently being investigated. HPLC assays for the determination of this analyte in human plasma and human urine have been developed. Isolation of I and the internal standard (II) was achieved by solid-phase extraction (SPE) in the 96-well format. A C₈ SPE plate was used for the extraction of the drug from human plasma (recovery >90%) while a mixed-mode (C₈/Cation) SPE plate was used to isolate the analytes from human urine (recovery approximately 71%). The analyte and internal standard were chromatographed on a Keystone Scientific Prism-RP^® guard column (20×4.6 mm) connected to a Prism-RP^® analytical column (150×4.6 mm), using a mobile phase consisting of 45% acetonitrile in 10 mM acetate buffer (pH=4); the analytes eluted at retention times of 5.2 and 6.9 min for I and II, respectively. Compounds I and II were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence (λ_ex=260 nm, λ_em=375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5–500 ng/ml for human plasma and human urine yielded a linear response (R²>0.99) when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the within-day precision for both assays was better than 7% C.V. at all points on the calibration curve; within-day accuracy was within 5% of nominal at all standard concentrations. The between-run precision and accuracy of the assays, as calculated from the results of the analysis of quality control samples, was better than 8% C.V. and within 8% of nominal. I was found to be stable in human plasma and urine for at least 8 and 2 months, respectively. In addition, the human plasma assay was semi-automated in order to improve sample throughput by utilizing a Packard liquid handling system and a Tom-Tec Quadra 96 SPE system. The precision and accuracy of the semi-automated procedure were comparable to the manual procedure. Over 5000 clinical samples have been analyzed successfully using these methods.     

Metabolic Behavior of O-Ethyl S,S-Diphenyl Phosphorodithiolate (edifenphos) in Female Goat

The determination of residues in fresh milk and the metabolic fate of edifenphos (O-ethyl S, S-diphenyl phosphorodithiolate), the active ingredient of the fungicide HINOSAN® were conducted in female goats using gas-liquid chromatographic techniques. Edifenphos was rapidly metabolized to form polar products which were excreted in the urine. The major metabolites found in the urine were O-ethyl S-phenyl hydrogen phosphorothiolate and O-conjugates of m- and p-(hydroxyphenyl) methyl sulfone. Several other metabolites; S, S-diphenyl hydrogen phosphorodithiolate, S-phenyl dihydrogen phosphorothiolate, O-ethyl dihydrogen phosphate, O-ethyl S-phenyl hydrogen phosphorodithiolate and methyl phenyl sulfone were also identified. In the feces, blood, some organs and tissues, edifenphos was scarcely detected. Oral administration of edifenphos to goat at the rate of 10 mg/kg resulted the secretion of 0.0008 ppm of edifenphos in milk as the maximum level after 6 hr of the administration.     

Metabolism of glucose,pyruvate, and glutamine during the maturation of oocytes derived from pre‐pubertal and adult cows

The aim of the study was to compare the energy metabolism of oocytes from pre‐pubertal (2 to 3 months) and adult cows during maturation, to identify the cause of poor developmental potential in many pre‐pubertal oocytes. The metabolism of [5‐³H] glucose, [2‐¹⁴C] pyruvate, and [G‐³H] glutamine was measured at 0 hr, 12 hr, and 24 hr maturation. Oxidative metabolism was important during maturation of oocytes from both pre‐pubertal and adult cows, with pyruvate metabolism peaking at 12 hr and glutamine metabolism increasing linearly and peaking at 24 hr. Peak oxidative metabolism was significantly lower in oocytes from pre‐pubertal animals, for both pyruvate and glutamine (P < 0.05). Glucose metabolism increased significantly during oocyte maturation in both groups (0hr to 24 hr). Glucose metabolism was significantly lower in oocytes from pre‐pubertal cows at 12 hr (P < 0.05). Oocytes from pre‐pubertal animals were significantly smaller than oocytes from adult cows at 0 hr, 12 hr, and 24 hr maturation (P < 0.05). When metabolic rates were corrected for oocyte volume, there were no significant differences in substrate metabolism between oocytes from pre‐pubertal and adult cows. There was however, a delay in the increase in glucose metabolism in pre‐pubertal oocytes 0 hr to 12 hr maturation. Germinal vesicle breakdown was slower in oocytes from pre‐pubertal animals with more oocytes still at the germinal vesicle stage approximately 5 hr post‐aspiration, compared to oocytes from adult cows (P < 0.05). By 24 hr, development to metaphase II was equivalent for pre‐pubertal and adult oocytes. This study identified differences in energy metabolism, oocyte size, and meiotic progression between the oocytes from pre‐pubertal and adult cows that may account for the poor developmental potential of many pre‐pubertal oocytes. Mol. Reprod. Dev. 54:92–101, 1999. © 1999 Wiley‐Liss, Inc.     

The metabolism of methylcyclohexane

1. When [U-¹⁴C]methylcyclohexane is fed to rabbits (dose 2–2·5m-moles/kg. body wt.), 65% of the radioactivity is excreted in the urine as metabolites, 0·5% appears in the faeces and about 15% in the expired air, some 4–5% remaining in the body in about 60hr. after dosing. The 15% of the dose appearing in the expired air consists of unchanged methylcyclohexane (10%) and ¹⁴CO₂ (5%). The low output of ¹⁴CO₂ shows that reactions leading to complete oxidation of methylcyclohexane are of minor importance. 2. The main metabolite found in the urine was the glucuronide of trans-4-methylcyclohexanol which was isolated. Seven methylcyclohexanols were found in the urine as conjugated glucuronides. The amounts of these were determined by isotope dilution to be as follows: cis-2-, 0·6%; trans-2-, 1·2%; cis-3-, 11·5%; trans-3-, 10·5%; cis-4-, 2·4%; trans-4-methylcyclohexanol, 14·7%, cyclohexylmethanol, 0·3%. No 1-methylcyclohexanol was found. There was evidence also that a small amount (approx. 1%) of the hydrocarbon aromatized to benzoic acid, probably via cyclohexylmethanol and cyclohexane-carboxylic acid. 3. The pattern of hydroxylation and the various amounts of the isomers found suggest that the hydroxylation in vivo of methylcyclohexane is dependent on steric factors in the molecule, hydroxylation occurring to the greatest extent at the carbon atom furthest away from the methyl group.     

Quantitation of o-, m- and p-cresol and deuterated analogs in human urine by gas chromatography with electron capture detection

A gas chromatographic method for the analysis of cresol metabolites of toluene and [²H₈]toluene in urine was developed. Cresol glucuronides and sulfates in urine were hydrolyzed with β-glucuronidase and arylsulfatase. Following extraction with tert.-butyl methyl ether and solvent exchange into benzene, the cresols were derivatized with heptafluorobutyric anhydride to form the heptafluorobutyrate esters. The derivatives were analyzed by gas chromatography with electron capture detection. Chromatographic resolution was achieved between all cresol isomers and their ²H₇ analogs. Calibration ranged from 0.001 to 500 μg/ml. Recoveries were 55–97% and showed no trend with respect to analyte concentration. Within-day precision of analyses of benchmark urine samples had a coefficient of variation of less than 4%. The assay sensitivity was limited by chromatographic background but was sufficient for quantification of the unlabeled cresols in urine from men with only environmental exposure to toluene. Average levels in urine samples from 45 men were 0.023, 0.054 and 37 μg/ml for o-, m- and p-cresol, respectively.     

Comparative analysis of the genomes of <Emphasis Type="Italic">Bombyx mandarina</Emphasis> and <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedroviruses

The Bombyx mandarina nucleopolyhedrovirus (BomaNPV) S1 strain can infect the silkworm, Bombyx mori, but is significantly less virulent than B. mori nucleopolyhedrovirus (BmNPV) T3 strain. The complete nucleotide sequence of the S1 strain of BomaNPV was determined and compared with the BmNPV T3 strain. The circular, double stranded DNA genome of the S1 strain was 126,770 nucleotides long (GenBank accession no. FJ882854), with a G+C content of 40.23%. The genome contained 133 potential ORFs. Most of the putative proteins were more than 96% identical to homologs in the BmNPV T3 strain, except for bro-a, lef-12, bro-c, and bro-d. Compared with the BmNPV T3 strain, however, this genome did not encode the bro-b and bro-e genes. In addition, hr1 lacked two repeat units, while hr2L, hr2R, hr3, hr4L, hr4R, and hr5 were similar to the corresponding hrs in the T3 strain. The sequence strongly suggested that BomaNPV and BmNPV are variants with each other, and supported the idea that baculovirus strain heterogeneity may often be caused by variation in the hrs and bro genes.     

Determination of ZD9583, a thromboxane receptor antagonist, in human plasma and urine by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

A liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry (LC–APCI-MS–MS) method is described for the determination of a thromboxane receptor antagonist (4Z)-6-((2S,4S,5R)-2-(1-(2-cyano-4-methylphenoxy)-1-methylethyl)-4-(3-pyridyl)-3-dioxan-5-yl)hex-4-enoic acid (ZD9583, I) in human plasma and urine. Proteins in plasma and urine samples are precipitated using acidified acetonitrile. The resulting supernatant is chromatographed on a C₈ reversed-phase chromatography column. Following the diversion of the solvent front from the mass spectrometer by a switching valve, the column eluate is passed on to the mass spectrometer via a heated nebulizer interface where the analyte is detected by multiple reaction monitoring (MRM). The method has a chromatographic run time of less than 2 min, a linear calibration curve with a range of 1–500 ng ml⁻¹ and intra- and inter-day precision estimates of less than 10% over the calibration range.