Spermine synthesis in the uterus of the ovariectomized rat in response to oestradiol-17β

We reported that spermidine and spermine pools in the uterus both doubled within 24h after oestradiol administration to castrated rats (Russell & Taylor, 1971). Now we have studied the enzymic synthesis of spermine (by spermidine-dependent S-adenosyl-l-methionine decarboxylase) and find that the activity of the enzyme(s) involved is elevated soon after hormone administration. Enzyme activity is increased within 4h and is five times that of controls within 24h. Cycloheximide or actinomycin D administered at the time of oestradiol injection completely blocked the increase in enzyme activity. The enzyme involved in spermine synthesis, S-adenosyl-l-methionine decarboxylase, with S-adenosyl-l-methionine and spermidine as required substrates, was partially purified on Sephadex and DEAE-cellulose columns. The decarboxylation of S-adenosyl-l-methionine could not be separated from the transfer of a propylamine moiety from the decarboxylated S-adenosyl-l-methionine to spermidine to form spermine. We were unable also to separate this system from the enzyme that formed spermidine when S-adenosyl-l-methionine and putrescine are used as substrates. Spermidine-stimulated S-adenosyl-l-methionine decarboxylase has an apparent half-life of 60min, identical with the half-life reported for putrescine-stimulated S-adenosyl-l-methionine decarboxylase. These results strongly suggest that the same enzyme(s) operate in the synthesis of both spermidine and spermine.     

Interferences of zearalenone,zearalanol or estradiol-17β with the steroid-metabolizing enzymes of the human prostate gland

In human hyperplastic prostate, 5α-dihydrotestosterone results from the 5α-reduction of testosterone and is transformed into epimeric 5α-androstanediols by the 3α- and 3β-hydroxysteroid dehydrogenases. Kinetics of these enzymic transformations were studied with labelled testosterone and 5α-dihydrotestosterone substrates in NADPH-supplemented homogenates from six hyperplastic prostate glands. Estradiol-17β interfered only with the prostatic 5α-reduction of testosterone. In contrast, other estrogenic substances found in food (zearalenone and zearalanol) inhibited only the 3α- and 3β-hydroxysteroid dehydrogenases. Increase in K_m in all cases and no statistically different increase of V_max pointed toward competitive types of inhibition. Since these in vitro studies only apply to NADPH-dependent enzymic activities, the inhibitory action of estradiol-17β on the prostatic 5α-reductase may occur in vivo. Nevertheless, effects of estradiol-17β and zearalenone or zearalanol on the NADH-dependent reductions of 5α-dihydrotestosterone and oxidations of the 5α-androstanediols need to be known before one of these compounds could be implicated in changes in levels of endogenous prostatic 5α-dihydrotestosterone.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17β and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes

1. The effects of graded doses of oestradiol-17beta and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [(14)C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10mug of oestradiol-17beta administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250mug of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10mug of oestradiol-17beta restored to the control value the inhibitory effect of a dose of either 50 or 125mug of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60-100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125mug of actinomycin D, administered to animals along with 10mug of oestradiol-17beta for 6-36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750mug of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17beta in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.     

Effect of 17β-estradiol on the human osteosarcoma cell line MG-63

We present evidence that 17β-estradiol (17β-E₂) regulates 1,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion by the human osteosarcoma cell line MG-63. When cells were pre-treated with 17β-E₂ for 48 h prior to treatment with 1,25(OH)₂D₃ (50 nM) for another 48 h, alkaline phosphatase activity increased by 40% (P < 0.025) with 2 nM 17β-E₂ and plateaued at levels of 20 and 200 nM 17β-E₂. Under the same experimental conditions, osteocalcin secretion was enhanced by 37% (P < 0.005) with 2 nM E₂. However, 17β-E₂ had no effect on basal alkaline phosphatase or on osteocalcin secretion. Moreover, simultaneous addition of 17β-E₂ and 1,25(OH)₂D₃ to cells did not result in any additional effect over l,25(OH)₂D₃ treatment alone. Tamoxifen (10 nM) inhibited 17β-E₂-induced activities in l,25(OH)₂D₃-treated cells while not affecting control cells. Dexamethasone pretreat-ment (100 nM, 48 h) also stimulated alkaline phosphatase activity in MG-63 cells. Moreover, dexamethasone pretreatment followed by treatment with 17β-E₂ and l,25(OH)₂D₃ gave an additive effect for alkaline phosphatase activity. 17α-Estradiol (17α-E₂), a less active form of estrogen, failed to modify, at low concentrations, control or l,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion. In fact, a 100–1000-fold higher concentration of 17α-E₂ was necessary to reproduce the effects of 17β-E₂ on osteocalcin secretion. The addition of insulin-like growth factor I (IGF-I) for 24 h (1–50 ng/ml) to MG-63 cells did not modify 1,25(OH)₂D₃-induced osteocalcin release from these cells. However, longer incubations with 50 ng/ml IGF-I did reproduce some of the effects observed with 17β-E₂. Thus, the effects of 17β-E₂ are probably not related to IGF-I production in MG-63 cells since under these conditions the addition of IGF-I alone should have produced a response at shorter incubation times and in the presence of lower concentrations of IGF-I. Since 17β-E₂ pretreatment was necessary to observe any effects on l,25(OH)₂D₃-induced activities, we hypothesized that 17β-E₂ regulated 1,25(OH)₂D₃ receptors in MG-63 cells. When cells were treated with 100 nM 17β-E₂ for 48 h, the binding affinity was unchanged: 37.3 ± 1.9 versus 35.1 ± 0.4 pM for cells whether treated or not with l7β-E₂, respectively. In contrast, a significant increase in binding capacity (B_max) was noted (15 ± 3.5%; P < 0.025). These results suggest that the estrogen analogue 17β-E₂ induces the differentiation of MG-63 cells into a more osteoblastic-like phenotype while 17α-E₂ is without physiological effect. They also suggest that estrogens may regulate bone remodeling by modulating hormonal-induction of proteins involved in bone mineralization. This effect is indirect since it does not modify basal activities, but involves a regulation of 1,25(OH)₂D₃ receptor levels in these MG-63 cells.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Changes in the patterns of synthesis of ribonucleic acid species in immature rat uterus in response to oestradiol-17β

1. After treatment of immature rats with diethylstilboestrol, the wet weight and RNA content of uterine tissue increased rapidly, reaching a peak at 40hr. After an initial lag of a few hours, the acid-soluble ribose and protein contents also rose to maxima at 40hr. No increase in DNA content occurred until at least 24hr. after treatment. 2. The RNA from immature rat uterus isolated at various times up to 6hr. after administration of oestradiol-17beta was labelled by injecting [(3)H]uridine and [(3)H]guanosine intraperitoneally 30min. before the animals were killed. It was fractionated on columns of kieselguhr coated with methylated serum albumin and the radioactivity in fractions corresponding to transfer RNA, 7s RNA, ribosomal RNA, Q(1)-RNA, Q(2)-RNA and DNA-like RNA was determined. 3. The radioactivity of the whole RNA increased steadily for 6hr. after hormone treatment. The earliest changes occurred in the Q(1)-RNA (ribosomal RNA precursor), whereas at longer time-intervals the radioactivity of the ribosomal RNA, 7s RNA and transfer RNA increased by four- to five-fold. The radioactivity of the DNA-like RNA increased by about 50%, but only at the longer time-intervals. 4. It is concluded that one of the earliest changes in response to oestradiol treatment is a major increase in synthesis of ribosomal RNA followed later by a similar increase in synthesis of transfer RNA and by a much smaller increase in synthesis of DNA-like RNA. The change in synthesis of ribosomal RNA in immature rat uterus may represent one of the most important responses to oestradiol treatment.     

The effect of zinc on the 5α-reduction of testosterone by the hyperplastic human prostate gland

The present studies were performed to evaluate the role of zinc in the regulation of testosterone 5α-reduction by the 800 g supematants prepared from human benign prostate hyperplasia specimens. The results show that when zinc is added at low concentrations the 5α-reduction of testosterone is increased but at higher cation concentrations the metabolism is significantly inhibited. This decrease was mediated by both a non-competitive inhibition of the binding of testosterone to the 5α-reductase enzyme and by a reduction in the formation of the NADPH cofactor. We have also demonstrated that the decreased synthesis of NADPH was produced by a competitive inhibition of both G6P and NADP binding to the G6PD enzyme. The data also suggests that the increase in testosterone metabolism observed at low zinc concentrations does not produce any changes in the binding of testosterone to the 5α-reductase enzyme. In spite of the above observations we were unable to establish any correlation between the endogenous zinc content of the tissue and the the in vitro capacity of the BPH samples to 5α-reduce testosterone. The present study suggests a possible physiological role for the regulation of testosterone metabolism by zinc in the human prostate gland.     

Regucalcin is expressed in rat mammary gland and prostate and down-regulated by 17β-estradiol

Regucalcin is involved in maintenance of calcium homeostasis due to the activation of Ca²⁺ pumping enzymes in the plasma membrane. It has a suppressive effect in cell proliferation, DNA and RNA synthesis, and may be associated with the abnormal cell division on tumor tissues. On the other hand both estrogens and Ca²⁺ are implicated in breast and prostate cancer but there are no studies focused on the expression of regucalcin in rat mammary gland or prostate. Furthermore, it is known that the expression of regucalcin in rat liver and kidney is regulated by 17β-estradiol (E₂). The aim of this study is to analyze if regucalcin is expressed in rat mammary gland and prostate and if it is regulated by E₂ in these tissues. We demonstrated for the first time that regucalcin mRNA and protein are present in rat mammary gland and prostate by in situ hybridization and immunohistochemistry, respectively. Furthermore, we show by Real-time PCR that E₂ down-regulates regucalcin expression in rat mammary gland and prostate.     

Synthesis of 2-substituted 17β-hydroxy/17-methylene estratrienes and their in vitro cytotoxicity in human cancer cell cultures

Synthesis of various types of 2-(alkylaminomethyl) and 2-(aroyl) 17β-estradiol analogs are reported. The synthesis of similar types of 2-substituted 17-methylene estratriene analogs was also achieved. Synthesis of chalcone derivatives of 17β-estradiol and 17-methylene estratriene were also realized. All these 2-substituted estratrienes were tested for their antiproliferative activity by using four different cell lines from colon, lung, glioma and breast cancers. Among the various 2-substituted estratrienes, the compounds 10d, 14a-h and 17e were found to have in vitro antiproliferative activity comparable to that of parent analogs 1-4. Comparison of the SAR pattern of these 2-susbtituted estratriene derivatives confirmed that relatively, 17-methylene estratrienes are more active than that of 17β-estradiol analogs.     

Effect of 17β-estradiol on cells stained for FSHβ and/or LHβ in the dog pituitary gland

Summary The effect of long-term treatment (52 weeks) with high doses of 17-estradiol (1.28 mg/kg/week intramuscularly) on gonadotrophs was studied in the pituitary gland of the beagle bitch. For immunochemical staining the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH were employed. For control purposes antisera to the following hormones were also used: bovine TSH, canine GH, canine PRL and porcine ACTH¹. In the pars distalis and pars tuberalis of control bitches, in addition to the cells which react solely with antisera to either LH or FSH, most cells were reactive to both antisera. The cells stained for FSH were less numerous than those shown to contain LH. TSH, PRL, GH and ACTH/MSH were localized in distinctly different cell types in the pars distalis of all control animals. In the treated bitches, almost complete regression of cells classically identified as gonadotrophs and stained for LH was observed. On the other hand, using the antiserum to FSH, selective immunochemical staining was localized in cells fitting the morphological characteristics of TSH cells. All these cells were also stained for TSH. However, a few cells were also shown to react solely with the antiserum to TSH. These cells, which seem to contain both TSH and FSH, were further clearly differentiated from PRL, GH and ACTH/MSH cells on the basis of their cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not necessarily apply to the glycoprotein hormones of the dog pituitary gland.Abbreviations of Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Mrs. K. Oertel for carrying out the experimental work on animals, to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17beta action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3mug or more of oestradiol-17beta. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2-2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17beta is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA.     

Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17beta given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [(14)C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17beta has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [(14)C]lysine into stromal nuclear proteins, but changes after oestradiol-17beta treatment were similar to those seen in epithelium with oestradiol-17beta alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17beta. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17beta alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-(3)H]oestradiol-17beta by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.     

Effect of anticancer drugs on the release of interferon γ in vitro

Summary Some conventional and experimental anticancer drugs were tested for their effect on concanavalin-A-induced interferon release from rat splenocytes in vitro. When 2.5 × 10⁶ rat splenocytes/ml, stimulated with 1 µg/ml concanavalin A, were incubated with various non-cytotoxic doses of the vinca alkaloid vincristine, there was an inhibition of the release of interferon in culture supernatants. The antitumour antibiotics bleomycin and Adriamycin, alkylating agents 4-hydroperoxycyclophosphamide and mafosfamide, and the immunoactive peptides FK 156 and FK565 did not affect the release of interferon under similar conditions. However, cyclosporin A, in similar experiments, markedly inhibited the release of interferon .     

Effect of thapsigargin on Ca²+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2?](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2?-sensitive fluorescent probe. Thapsigargin at concentrations between 10?nM and 10 μM increased [Ca2?](i) in a concentration-dependent fashion. The Ca2? signal was reduced partly by removing extracellular Ca2? indicating that Ca2? entry and release both contributed to the [Ca2?](i) rise. This Ca2? influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2? channels or by modulation of protein kinase C activity. In Ca2?-free medium, pretreatment with the endoplasmic reticulum Ca2? pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2? release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2?](i) rise, suggesting that thapsigargin released Ca2? from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca2?](i) rise. At concentrations of 1-10 μM, thapsigargin induced cell death that was partly reversed by chelation of Ca2? with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca2?](i) rises by causing phospholipase C-independent Ca2? release from the endoplasmic reticulum and Ca2? influx via phospholipase A2-sensitive Ca2? channels. Thapsigargin also induced cell death via Ca2?-dependent pathways and Ca2?-independent apoptotic pathways.     

β-Glucanase specific expression in the parotid gland of transgenic mice

The feasibility of using the pig parotid secretory protein promoter to drive the β-glucanase transgene expression in mouse parotid glands was examined in this study. The parotid gland-specific vector expressing β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was constructed. Transgenic mice were produced by the pronuclear microinjection. Both PCR and Southern blot analysis showed that the mice carried the β-glucanase gene and the β-glucanase gene could be stably inherited. Furthermore, RT-PCR and northern blot analysis indicated that it was specifically expressed in the parotid. The β-glucanase activity in the saliva was found to be 0.18 U/mL. After feeding a diet containing 2 % β-glucan, the average daily gain of transgenic was significantly higher than non-transgenic mice. The crude protein and crude fat concentration in faeces of transgenic mice were significantly reduced compared with that of the non-transgenic mice. These results suggest that the successful expression of foreign β-glucanase in the animal parotid would offer a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.     

Effect of activated charcoal and pruning of the taproot on the in vitro mycorrhization of Hevea brasiliensis Müll. Arg.

In vitro mycorrhization of Hevea brasiliensis under autotrophic culture conditions is a promising methodology to produce plantlets adapted to overcome stresses during acclimatization. However, to succeed in the in vitro production of mycorrhizal plantlets, root production and subsequent colonization by the mycorrhizal fungus need to be optimized. Plantlets of H. brasiliensis clone PB 260 were grown in contact with the extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833. Addition of activated charcoal to the medium and pruning of the taproot were evaluated for their effects on root growth and colonization. None of the treatments stimulated the early formation of new roots. However, total root length, total root colonization, and production of arbuscules and intraradical spores/vesicles were significantly higher in plantlets grown in the presence of activated charcoal (especially after 13 wk of culture). In contrast, total root colonization was significantly lower in the pruned plantlets, while total root length and arbuscule formation were not affected. None of the treatments affected activities of succinate dehydrogenase and alkaline phosphatase measured in the extraradical mycelium of the fungus. It appeared that the addition of activated charcoal to the culture medium favored root growth and mycorrhization of rubber plantlets under in vitro culture conditions, while taproot pruning did not favor these parameters.