Estrogenic activity of zearalenone and zearalanol in the neonatal rat uterus

Fusarium sp. contaminated feedstuffs elicit adverse estrogenic effects in several commercially important animal species via the mycotoxin zearalenone. An estrogenically active synthetic derivative, zearalanol, is used as an anabolic agent in cattle. Since estrogens can irreversibly alter target tissue development, we investigated the estrogenic activity of these compounds in the neonatal rat uterus. Both induced dose-dependent premature uterine growth when injected daily on postnatal days 1-5 (ED50 = 1.3 mg/kg BW). Nuclear estrogen receptor levels dramatically increased 1 hour after either a single injection on day 5 or after five daily injections. In 5-day-old animals, the translocated nuclear receptor was characterized as a single class of binding sites with a dissociation constant (KD) for estradiol (E2) of 1 nM. At 15 days, zearalanol-treated animals showed greater uterine nuclear receptor retention than zearalenone-treated animals. In 5-day-old animals, single mycotoxin doses induced five fold elevations of ornithine decarboxylase (ODC) at 6 hours. Unlike the growth response, ODC dose-response studies showed zearalanol to be about 20-fold more effective than zearalenone. Time course studies revealed that a low dose of zearalenone, but not of zearalanol, resulted in a shift in peak activity from 6 to 8 hours. These data suggest that metabolism of zearalenone may be important in short-term pharmacodynamics. In a competitive binding assay, neither compound competed [3H]E2 from the E2 binding site on alpha-fetoprotein. We conclude that the uterine growth response and ODC induction demonstrate the neonatal estrogenic action of these mycotoxins, apparently mediated via the estrogen receptor. The greater effectiveness of zearalanol in inducing ODC may be related to nuclear retention and/or zearalenone metabolism.     

Radioimmunoassay of haloperidol in human serum

Immunization of rabbits with a haloperidol hydrazone-bovine serum albumin conjugate elicited the formation of antibody to haloperidol. With this antiserum, concentrations of haloperidol as low as 1 ng/ml can be easily measured by radioimmunoassay of unextracted human serum. None of the known major metabolites of haloperidol displayed any significant cross-reactivity. Psychiatric patients receiving daily oral doses of 20 to 200 mg haloperidol had serum levels ranging between 5.8 and 245 ng/ml.     

Radioimmunoassay of melatonin: human serum and cerebrospinal fluid

Using a newly characterized anti-melatonin serum it has been possible to establish human serum melatonin concentrations at short time intervals during 24h. A clear circadian rhythm with peak values during the dark phase was demonstrated in both men and women. Values (pg/ml;mean +/- SE) were as follows: females 02.00h: 130 +/- 7, 18.00h: 45 +/- 5 males 02.00h: 140 +/- 11, 18,00H: 70 +/- 5. The estimation (pg/ml; mean +/- SE) of melatonin in human serum (males: 63 +/- 22 and females: 100 +/- 45) and cerebrospinal fluid simultaneously taken has shown that melatonin is lower in cerebrospinal fluid (males: 59 +/- 33 and females: 57 +/- 28). Blanks are not subtracted.     

Radioimmunoassay for estimation of thyroglobulin in human serum.

A specific double antibody radioimmunoassay has been develop for the measurement of thyroglobulin in human serum. Human thyroglobulin was purified by combined DEAE-cellulose and affinity chromatography using Sepharose 4B-bound Concanavalin A. Sensitivity of test serum was 10 ng/ml. Thyroglobulin was not detectable in half of normal subjects, and half showed values between 10 and 180 ng/ml. In the patients with simple goiter and secondary hypothyroidism, serum thyroglobulin was usually in the normal range. In Hashimoto's thyroiditis, many sera having precipitating antibodies or high hemagglutination antibodies for thyroglobulin showed a high thyroglobulin concentration in serum probably due to a false positive reaction. In hyperthyroidism, an increased thyroglobulin level was observed in 64% of patients. However, there was no correlation between serum thyroglobulin and thyroxine levels in untreated hyperthyroidism. Serum thyroglobulin was increased significantly in some cases for several weeks after isotope therapy for the hyperthyroidism.     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Radioimmunoassay for N6(methylnitroso)adenosine: production and characterization of antibodies

Antibodies specific for N⁶(methylnitroso)adenosine have been produced in rabbits and a sensitive radioimmunoassay was developed. The nitroso group is immunodominant; 50% inhibition of the binding of [³H]N⁶(methylnitroso)adenosine to antibody was obtained with 9.6 pmoles of N⁶(methylnitroso)adenosine and 200 nmoles of N⁶-methyladenosine. Adenosine was essentially inactive. After nitrosation, N⁶(methylnitroso)adenosine can be detected only in those RNA molecules known to contain N⁶-methyladenosine.     

Radioimmunoassay of haloperidol in human serum: Correlation of serum haloperidol with serum prolactin

A radioimmunoassay (RIA) for measurement of serum haloperidol is described. Compared to gaschromatography (GC), RIA values average 40% higher. However, a simple organic extraction of serum yields statistically equivalent RIA and GC haloperidol determinations. For both men and women combined, there was a positive correlation between dose (mg/kg/day) and steady-state serum haloperidol level (r = +0.86) and between steady-state serum haloperidol and serum prolactin (PRL) concentration (r = +0.87).     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies.

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

A simple method for obtaining transferrins from human plasma and porcine serum: preparations and properties

A simple method was described for the purification of serum transferrin (Tf) from human plasma and porcine serum with relative high yield and purity. The properties including purity, integrity, immunoreactivity and the receptor-binding ability of the proteins were studied by several assays, comprising spectrometry, SDS-PAGE, HPLC, Western blotting, urea electrophoresis, mass spectrometry and cytometry. Analysis from all the different aspects manifested that the proteins were of high purity. The two kinds of Tfs appeared to be iron-saturated as confirmed by their absorbance spectra and urea-PAGE mobility. The specific spectra of absorption of the two Tfs were both at around 465 nm. The relative molecular weights of human Tf (hTf) and porcine Tf (pTf) were determined by SDS-PAGE and further identified by MAIDI-TOF mass spectrometry with a result of 79,707 and 79,258, respectively. Immunoblotting assay showed that pTf could react with the anti-human Tf monoclonal antibody with a less level compared to hTf. FACS assays of their binding activities to Tf receptor-positive cell (K562 cell line) indicated that pTf could be recognized by the hTf receptor and internalized into cells, with a slightly less efficacy than hTf. All special property studies demonstrated that pTf was similar to hTf in physical and chemical characteristics, which gave a hint that pTf could substitute for hTf in some kinds of researches, such as using hTf as a carrier in drug targeting system.     

Production and analytical properties of antibodies with high specificity to zearalenone

The time course of production, specificity, and analytical potential of anti-zearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; alpha-zearalenol, 0.15%; and beta-zearalenol, < 0.02%. With synthetic analogues: zearalanone, 31.7% and alpha-zearalanol, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.     

Radioimmunoassay without extraction of L-triiodothyronine in human serum]

A radio-immunoassay of L-triiodothyronin is described. Blinding of T3 to TBG is prevented by salicylate (1%--w/v). The rabbit antiserum obtained by injection of a complexe T3-bovine serum albumin is diluted between 1/10000 and 1/20000. Bound and free hormone are separated by polyethylene glycol (17%--w/v). Serum T3-concentration in normal subjects averaged 132 +/- 29 ng/dl (SD). In hyperthyroid and hypothyroid patients, these values were respectively 351 +/- 118 ng/dl and 68 +/- 21 ng/dl.     

Radioimmunoassay for antibodies in varicella-zoster virus serology.

The method of RIA for antibodies was employed with success in VZ virus serology. The method is suitable for testing VZ antibodies in the course of varicella or herpes zoster disease as well as for determining anamnestic titres. Its advantages are stability of antigen, objective reading of results and applicability to testing large serum sets.     

Fungicide inhibition of aflatoxins,diacetoxyscirpenol and zearalenone production

The effect of fungicides on the production of aflatoxin byAspergillus flavus IMI 89717, diacetoxyscirpenol and zearalenone byFusarium graminearum was studied. In a yeast extract - sucrose medium, dicloran, iprodione and vinclozolin fungicides significantly inhibited mycelial growth ofA. flavus at 250 ppm and significantly decreased aflatoxin production at 100, 250 and 500 ppm, respectively. In potato — dextrose broth, these fungicides diminished the mycelial growth ofF. graminearum and production of diacetoxyscirpenol and zearalenone at 100 ppm. Sensitivity of toxigenic mycelia to fungicides increased approximately five-fold in a yeast extract-starch medium with an appreciable reduction in sugar uptake andα-amylase activity.     

Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum

Applied Microbiology and Biotechnology - The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system,...