Application of avidin-ferritin and peroxidase as contrasting electron-dense markers for simultaneous electron microscopic immunocytochemical labelling of glutamic acid decarboxylase and tyrosine hydroxylase in the rat arcuate nucleus

Summary A pre-embedding immunostaining procedure was developed using ferritin and peroxidase to enable simultaneous electron microscopic localization of two antigens in the same tissue section. This method was used to study the anatomic relationship between glutamic acid decarboxylase (GAD) immunoreactive axons and tyrosine hydroxylase (TH) — containing neurons of the rat arcuate nucleus. The findings provide ultrastructural evidence that GAD-immunoreactive terminals establish symmetric (Gray II) synapses on TH-reactive neurons.     

Estrogen hydroxylase activity in the human placenta at term

Placental estrogen hydroxylase (EH) enzyme activity was measured at term using the catechol-O-methyl transferase coupled method in normal and high risk conditions. The identity and ratio of products formed during incubation of microsomes as analysed by high performance liquid chromatography in chronic hypertension, toxemia and diabetes mellitus was not different from controls. The mean enzymatic activity was also not different among the conditions studied as expressed mean +/- SE pmol/min/mg, protein: chronic hypertension (7.8 +/- 1), toxemia (8 +/- 1.6), diabetes mellitus (6.1 +/- 0.9) and controls (8.3 +/- 1.5). The cofactor dependence of EH was studied showing that NADPH is a better substrate for the enzyme than NADH.     

The use of gold-substituted silver-intensified diaminobenzidine (DAB) and non-intensified DAB for simultaneous electron microscopic immunoperoxidase labeling of tyrosine hydroxylase and glutamic acid decarboxylase immunoreactivity in the rat medial preoptic area

An improved gold-substituted silver intensification procedure for the peroxidase-diaminobenzidine (DAB) reaction product was developed. The method was applied in the rat medial preoptic area to label tyrosine hydroxylase (TH)-immunoreactive profiles. Following the gold toning, the same sections were immunostained for glutamic acid decarboxylase (GAD) immunoreactivity with non-intensified peroxidase-DAB. Single DAB-labeled GAD axons were found in symmetric synaptic connection with unlabeled dendrites as well as with gold-toned immunoperoxidase-containing TH neurons.     

Effect of 5 alpha-dihydrotestosterone on sexual receptivity and neural progestin receptors in ovariectomized rats given pulsed estradiol

Experiments were carried out to examine the mechanism whereby 5 alpha-dihydrotestosterone (DHT) antagonizes the stimulatory effects of estrogen plus progesterone (P) on sexual receptivity (lordosis) in the ovariectomized rat. Estradiol (E2; 1 microgram s.c. in 10% ethanol) was administered in a discontinuous (pulsed) treatment regimen thought to mimic phase requirements of estrogen action; two injections of E2 were given either 6 or 12 h apart (first injection, Hour 0). Progesterone (0.5 mg in oil) was injected at Hour 20, and behavioral testing occurred at Hour 24. Dihydrotestosterone (2.5 mg s.c. in 10% ethanol/propylene glycol) inhibited lordosis when it was given before (-12 or -3 h), between (+3, or -3 and +3 h), or after (+8 h) the two E2 injections, but was not effective when given at +20 h. Significant inhibition of E2 + P-induced lordosis was achieved by 2.5 but not 1.0 or 0.2 mg DHT at -3 h, while uterine weights in the same animals were reduced significantly by 2.5 and 1.0 mg DHT. Serum E2 and DHT concentrations peaked rapidly after injection, declining to near baseline by 3 and 12 h, respectively. Induction of cytosolic progestin receptors (cPR) in the preoptic area and medial basal hypothalamus by estrogen was not prevented by DHT when animals were given the two pulsed E2 injections or daily injections of estradiol benzoate, although P was able to override the inhibitory behavioral effects of DHT in the latter but not the former group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of catechol estrogen-estrogen receptor dissociation: a possible factor underlying differences in catechol estrogen biological activity

The mechanisms underlying the differences in uterotrophic potency between 2- and 4-hydroxyestrogens were explored. Doses of estradiol (E2)(10 micrograms/kg), 2-OHE2 (500 micrograms/kg) and 4-OHE2 (100 micrograms/kg) sufficient to induce near maximal cell nuclear estrogen receptor (ERn) binding were injected subcutaneously into 26 day old female rats. Uterine ERn concentrations declined more rapidly after 2-OHE2 than after E2 or 4-OHE2. E2 and 4-OHE2 both elicited a significant increase in uterine wet weight, measured at 24-36 hrs after injection. 2-OHE2 had no significant effect and neither synergized with nor antagonized the effects of simultaneously administered E2 or 4-OHE2. Under in vitro conditions at 25 degrees C, 2-hydroxyestrone (2-OHE1) and 2-OHE2 both dissociated from the receptors more rapidly than either their parent monophenolic estrogens or the corresponding 4-hydroxyestrogens. These results suggest that differences in estrogenic potency between 2- and 4-hydroxyestrogens may partly be a function of the dissociation kinetics of their estrogen receptor complexes.     

The synthesis and testing of E-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone and Z-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone as gamma-emitting ligands for the androgen receptor

Two iodinated steroids, E-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone and Z-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone were synthesized in a search for a gamma-emitting androgen that binds with high affinity to the androgen receptor. Such compounds would be extremely useful research tools for studies of androgen responsive tissues and as in vivo probes of androgen responsive tumors such as prostate cancer. These 17 alpha-iodovinyl steroids were synthesized because many 17 alpha-substituents do not interfere markedly with binding to the androgen receptor and because similar analogs of other steroids, estrogens and progestins, have been shown to have the requisite properties for ligands to those receptors. Both of these potential ligands were tested for their ability to compete with [3H]R1881 for binding to the androgen receptor in cytosols from prostate, hypothalamus and pituitary. The relative binding affinities ranged between 5 and 20%, depending upon the tissue and steroid. In order to test the two ligands directly, they were both synthesized labelled with 125I and tested for binding to the androgen receptor in prostatic cytosol and in vivo for specific concentration in androgen responsive tissues. While there was considerable binding in the prostatic cytosol, it was not specific because 5 alpha-dihydrotestosterone did not compete. Likewise in the in vivo experiment there was no evidence for androgen receptor mediated concentration of the tracers. While on the basis of relative binding affinity, these 2 steroids appeared to be good candidates for androgen receptor ligands, neither were useful for this purpose. These results contribute new information which will be valuable in the design of other gamma-emitting androgens and emphasises that, in this process, other factors such as metabolism and nonspecific binding must be considered.     

Separation of steroidal estrogens and their major unconjugated metabolites by high performance liquid chromatography

A high performance liquid chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedures uses a "Diol" column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone, one of the least polar C18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semi-preparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Attenuating the defeminization of the neonatal rat brain: Mechanisms of action of cyproterone acetate, 1,4,6-androstatriene-3,17,-dione and a synthetic progestin, R5020

Although defeminization of the rat brain appears to depend significantly on the conversion of testosterone (T) to estradiol (E₂), the antiandrogenic steroid cyproterone acetate (CA) is able to attenuate defeminization. In order to study the mechanism of action of CA on brain sexual differentiation, newborn male rats were given subcutaneous injections of this steroid on postnatal Days 2–6. When castrated on Day 70 and given estrogen and progesterone, these CA-treated males displayed elevated lordosis quotients (LQ) compared to controls. CA-treated neonatal males were also examined at the end of the drug treatment to ascertain the mechanism of drug action: (1) Serum T levels were normal; (2) Brain cell nuclear estrogen receptor occupation, estimated by an exchange assay, was reduced by ≈ 30% in the brains of the CA-treated males, although the ability of exogenous E₂ to occupy these brain estrogen receptors was not reduced. Other work has demonstrated a weak competitive effect of cyproterone on aromatization, and thus cyproterone acetate may have interfered with the conversion of T to E₂ CA also has progestogenic activity, and 5-mm capsules of a potent synthetic progestin, R5020, given to newborn male rats on Days 2–6, are shown to elevate the LQ after postnatal Day 70 to the same extent as CA. However, R5020 did not reduce estrogen receptor occupation in the neonatal male rat brain and was without effect on serum T levels in the neonatal male. Because of the implied role of T-derived estrogens in defeminization, an experiment was conducted showing that the defeminizing action of estradiol benzoate given to 3-day-old female rat pups is attenuated by the antiestrogen, CI628, and not by the potent inhibitor of aromatization, 1,4,6-androstatriene-3,17-dione (ATD). This result complements previous experiments showing that both ATD and CI628 attenuate the defeminization produced by T. Taken together, the results lend further support to a pivotal role for aromatization and for estrogen-receptor interactions in the defeminizing effects of T. The actions of progestins such as CA and R5020 in attenuating defeminization are discussed in relation to the recent demonstration of progestin receptors in the neonatal rat brain. It is concluded that CA may act by a combination of actions, both by inhibiting aromatization and by acting as a progestin.