Theoretical Models of Chronotherapy: I. Periodic Perturbations in Oscillating Chemical Reactions

Dynamics resulting from periodic perturbations in oscillating chemical reaction, as theoretical models of chronotherapy techniques are studied. Replication reactions and fluctuations take place in these models giving rise to new species with autocatalitic properties. Oscillating chemical reaction models due to Hanusse (H) with three intermediaries and Bourceanu (E₁, E₂, E₃ ) with four, five and six intermediaries, respectively, are taken for this study and are manipulated numerically using a Gear algorithm. The bifurcation diagrams are obtained applying non-linear dynamic techniques, such as, power spectrum, stroboscopic method, Lyapunov exponents and correlation dimension D 2. The dynamics of Hanusse and Bourceanu perturbed models are very similar. The main dynamic behaviours observed after perturbation are quasiperiodicity, entrainment, some Arnold tongues and situations without chemical meaning. Chaos is not observed. This study contributes to understand theoretical and practical aspects of the chronotherapy techniques.     

蝉虫草样品的分级萃取与化学成分分析

利用索氏萃取技术,依次采用石油醚、乙酸乙酯、丙酮、无水乙醇和甲醇等5种溶剂对蝉虫草纯粉进行分级萃取,运用傅里叶变换红外光谱分析法和气相色谱-质谱联用技术对5级萃取物进行分析鉴定。傅里叶变换红外光谱分析显示萃取物中含有与烯烃类、羧酸类、酯类、醇类和酮类等化合物相关的C-H、C=O、C-O和C=C等官能团。气相色谱-质谱联用技术共鉴定出有机小分子化合物34种,以酯类和脂肪酸类为主,多为碳链长度为15-20的长链脂肪酸及对应的酯,其中十八碳不饱和脂肪酸相对含量高达28.95%;分别存在于两种或以上萃取物中的有机化合物共有11种;仅存于石油醚萃取物中的化合物6种,乙酸乙酯萃取物中3种,丙酮萃取物中2种,无水乙醇萃取物中6种,甲醇萃取物中6种。在一定极性范围内,利用溶剂的极性梯度变化,可实现蝉虫草中活性物质的按极性梯度分离;采用分级萃取技术可有效分离蝉虫草中部分化学成分。鉴定结果充实了蝉虫草中化合物的种类资源,为蝉虫草中活性物质谱图库的完善、构效关系的建立及蝉虫草产品的利用开发提供支撑。     

Estrogen effects on NADH oxidase and superoxide dismutase in prepubertal female rats

Thirty-four day old, ovariectomised rats were treated with increasing doses of estradiol, 2-hydroxyestradiol 2,3-dimethyl ether (23E₂), 4-hydroxyestradiol 3,4-dimethyl ether (34E₂) and 4-methoxy-estradiol (4ME₂) for five days by subcutaneous injection. Superoxide dismutase, phenol activated NADH oxidase and uterine dry weights were determined. Only estradiol was found to be uterotrophic and increased NADH oxidase activity in these experiments. Both 23E₂ and 34E₂ treatment reduced the enzyme activity significantly. Though 4ME₂ showed a decrease in NADH oxidase at 0.05μg/100gm body weight there was no further decrease at higher dose (5μg/100gm). The Superoxide dismutase (SOD) in uterus and liver was unaffected by estradiol, while 23E₂, 34E₂, and 4ME₂ significantly reduced SOD in both liver and uterus. These results indicate that 23E₂, 34E₂ and 4ME₂, in spite of their non-uterotrophic property, affect uterine metabolism. Furthermore, in view of the reports indicating the importance of SOD levels in various tumors and since catecholestrogens are observed to reduce SOD levels in liver and uterus, it is suggestive that catecholestrogens may play an important role in the pathophysiology of certain tumors.     

The membrane effects of 17beta-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.     

Estrone sulfate-sulfatase and 17β-hydroxysteroid dehydrogenase activities: a hypothesis for their role in the evolution of human breast cancer from hormone-dependence to hormone-independence

The evaluation of estrogens (estrone, estradiol, and their sulfates) in the breast tissue of post-menopausal patients with breast cancer indicates high levels, particularly of estrone sulfate (E₁ S) which is 15–25 times higher than in the plasma. Breast cancer tissue contains the enzymes necessary for local synthesis of estradiol and it was demonstrated that, despite the presence of the sulfatase and its messenger in hormone-dependent and hormone-independent breast cancer cells, this enzyme operates particularly in hormone-dependent cells. Different progestins: Nomegestrol acetate, Promegestone, progesterone, as well as Danazol, can block the conversion of E₁ S to E₂ very strongly in hormone-dependent breast cancer cells. The last step in the formation of estradiol is the conversion of E₁ to this estrogen by the action of 17β-hydroxysteroid dehydrogenase. This activity is preferentially in the reductive direction (formation of E₂) in hormone-dependent cells, but oxidative (E₂ → E₁) in hormone-independent cells. Using intact hormone-dependent cells it was observed that Nomegestrol acetate can block the conversion of E₁ to E₂. It is concluded, firstly, that in addition to ER mutants other factors are involved in the transformation of hormone-dependent breast cancer to hormone-independent, this concerns the enzymatic activity in the formation of E₂; it is suggested that stimulatory or repressive factor(s) involved in the enzyme activity are implicated as the cancer evolves to hormone-independence; secondly, different drugs can block the conversion of E₁ S to E₂. Clinical trials of these “anti-enzyme” substances in breast cancer patients could be the next step to investigate new therapeutic possibilities for this disease.     

Stimulatory and inhibitory effects of dimethylsulfoxide, propranolol and chlorpromazine on the partial reactions of ATPase of sarcoplasmic reticulum

The effects of dimethylsulfoxide, propranolol and chlorpromazine on the partial reactions of the ATPase of sarcoplasmic reticulum were investigated. When analyzed according to a reaction scheme in which the ADP-sensitive (E₁P) and ADP-insensitive (E₂P) phosphoenzymes occur sequentially and P_i is derived from the latter, dimethylsulfoxide decreased the rate of E₂P hydrolysis whereas it stimulated the rate of the E₁P to E₂P conversion. Propranolol increased the rate of E₂P hydrolysis while it decreased the rate of the E₁P to E₂P conversion. Propranolol exerted an additional effect, presumably inhibition of the phosphoenzyme formation. These effects of dimethylsulfoxide and propranolol can account for both the stimulatory and inhibitory effects of these drugs on the overall rate of ATP hydrolysis observed in the presence and absence of added alkali metal salts.

Chlorpromazine accelerated E₂P hydrolysis whereas it appeared to inhibit the E₁P to E₂P conversion. These effects of chlorpromazine appear able to account for its stimulatory and inhibitory effects on the overall rate of ATP hydrolysis in the presence and absence of alkali metal salts. In the presence of chlorpromazine, however, the rate of P_i liberation during the steady state ATP hydrolysis was found to be greater than the hydrolysis rate of E₂P. This finding suggests that under these conditions P_i is derived not only from E₂P but also from source(s) other than E₂P.     

Estrogen metabolism and formation of estrogen-DNA adducts in estradiol-treated MCF-10F cells. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction and catechol-O-methyltransferase inhibition

Formation of estrogen metabolites that react with DNA is thought to be a mechanism of cancer initiation by estrogens. The estrogens estrone (E₁) and estradiol (E₂) can form catechol estrogen (CE) metabolites, catechol estrogen quinones [E₁(E₂)-3,4-Q], which react with DNA to form predominantly depurinating adducts. This may lead to mutations that initiate cancer. Catechol-O-methyltransferase (COMT) catalyzes an inactivation (protective) pathway for CE. This study investigated the effect of inhibiting COMT activity on the levels of depurinating 4-OHE₁(E₂)-1-N3Ade and 4-OHE₁(E₂)-1-N7Gua adducts in human breast epithelial cells. MCF-10F cells were treated with TCDD, a cytochrome P450 inducer, then with E₂ and Ro41-0960, a COMT inhibitor. Estrogen metabolites and depurinating DNA adducts in culture medium were analyzed by HPLC with electrochemical detection. Pre-treatment of cells with TCDD increased E₂ metabolism to 4-OHE₁(E₂) and 4-OCH₃E₁(E₂). Inclusion of Ro41-0960 and E₂ in the medium blocked formation of methoxy CE, and depurinating adducts were observed. With Ro41-0960, more adducts were detected in MCF-10F cells exposed to 1 μM E₂, whereas without the inhibitor, no increases in adducts were detected with E₂ ≤ 10 μM. We conclude that low COMT activity and increased formation of depurinating adducts can be critical factors leading to initiation of breast cancer.     

An optimised, highly sensitive radioimmunoassay for the simultaneous measurement of estrone, estradiol and estrone sulfate in the ultra-low range in human plasma samples

Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E₁), estradiol (E₂) and estrone sulfate (E₁S) levels in the ultra-low range. Following incubation with [³H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E₁S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E₁, E₂) were separated by chromatography (LH-20). Estrone and E₁S (following hydrolysis) were converted into E₂, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[¹²⁵I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E₁, E₂ and E₁S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E₁, 0.67 pmol/L for E₂, and 0.55 pmol/L for E₁S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E₁, 5.1% for E₂ and 6.1% for E₁S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E₁, E₂, and E₁S, respectively. Considering normal plasma levels for E₂ (15 pmol/L), E₁ (80 pmol/L) and E₁S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E₂, E₁ and E₁S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels.     

Evidence for the DNA binding and adduct formation of estrone and 17β-estradiol after dimethyldioxirane activation

Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E₁) and 17β-estradiol (E₂) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957–1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E₁ and E₂ after activation are able to bind DNA. This paper reports that after DMDO activation, [³H]E₁ and [³H]E₂ were able to bind to both A-T and G-C containing DNAs. Furthermore, the formation of E₁–DNA and E₂–DNA adducts was detected by ³²P-postlabeling analysis.     

Reversal of tamoxifen resistant breast cancer by low dose estrogen therapy

Currently, the standard of care for estrogen receptor (ER)-positive breast cancer is 5 years of tamoxifen (TAM) or an aromatase inhibitor (AI) such as anastrozole. New studies indicate that extending antiestrogen therapy beyond 5 years with sequential regimens will improve disease-free survival. Based on the emerging concept that longer therapies are better, we have developed sequential models of tamoxifen-resistant breast cancer in vivo to mimic the clinical scenario of long-term antiestrogen therapy. The goal of the current study was to investigate the consequences of long-term treatment with tamoxifen on the growth of breast tumors in athymic mice. The results demonstrate that there are distinct phases of resistance to tamoxifen that correlate with time of treatment and expression of HER2/neu mRNA. In the treatment phase, 17β-estradiol (E₂) stimulated growth, while TAM inhibited growth of MCF-7 tumors (MCF-7E₂). The withdrawal of treatment, mimicking the use of an AI, completely prevented growth. In Phase I resistance, the tumors (MCF-7TAMST) were growth-stimulated by either E₂ or TAM, but inhibited by no treatment, fulvestrant, or E₂ + fulvestrant. Phase II-resistant tumors (MCF-7TAMLT) were treated for more than 5 years and growth-stimulated by TAM. However, no treatment, fulvestrant, or E₂ completely inhibited growth. Interestingly, the few tumors (MCF-7TAMLT) that survived in response to E₂ were robustly re-stimulated by E₂ after transplantation into new generations of athymic mice. These E₂-stimulated tumors (MCF-7TAME) were inhibited by TAM in a dose-dependent similar to their parental tumors (MCF-7E₂). In addition, the MCF-7TAME tumors were inhibited by either no treatment or fulvestrant. HER2/neu and HER3 mRNAs were over-expressed in TAM-stimulated MCF-7TAMLT tumors and remained high in E₂-stimulated MCF-7TAME tumors. The data indicate that complete reversal of resistance to TAM can be achieved with the use of low dose E₂ therapy. Also, these data suggest that over-expression of HER2/neu alone is insufficient to predict resistance to TAM. Based on the results, we suggest using an alternating treatment regimen, cycling antiestrogen with estrogen therapy to avoid drug-resistance.     

A Monoclonal Antibody to Oestradiol Potentiates the Stimulation of the Specific Activity of the Brain Type Creatine Kinase by Oestrogen in vivo and in vitro

We described previously the in vivo immunoneutralization effects of a high affinity anti-oestradiol antibody clone 15 in blocking ovulation and synaptic remodeling in cycling female rats. In the present study we report the enhancing effects of this antibody. Treatment of ovariectomized female rats or female derived skeletal cell cultures in vitro with anti-E₂ 15 plus oestrogen (E₂) potentiated the specific activity of the brain type creatine kinase (CK) response to E₂ in the rat tissues or skeletal cells. The enhancing CK response of anti E₂ 15 plus E₂ was time- and dose-dependent in the uterus, thymus, epiphysis and diaphysis of ovariectomized female rats. In the pituitary, on the other hand, anti-E₂ 15 blocked the stimulatory CK response to E₂. Two other high affinity anti-E₂ anti-bodies, clones 8D₉ and 11B₆, had no effect in augmenting the response of CK to E₂ in rat tissues. Moreover, the enhancing CK response in rat tissues was specific to anti-E₂ 15 plus E₂ since the intact anti-E₂ in the presence of other oestrogen mimetics, such as oestriol or stilbestrol or tamoxifen did not potentiate the CK response in rat tissues. In this model system the Fab' monomer of anti-E₂ 15 abolished the CK response to E₂ in rat tissues and not to anti-E₂ 15 plus E₂ whereas tamoxifen completely blocked the CK response to anti E₂ plus E₂. Anti E₂ 15 may therefore serve as a specific carrier in delivering E₂ to oestrogen sensitive rat tissues or cells containing functional oestrogen receptors and thereby increasing the magnitude of E₂ effects in vivo and in vitro.     

17β-Hydroxysteroid dehydrogenase activity in endometrial cancer cells: Different metabolic pathways of estradiol in hormone-responsive and non-responsive intact cells

In this paper we report that two human long-term endometrial cancer cell lines, Ishikawa and HEC-1A, exhibit quite different abilities in metabolizing estrogens. As a matter of fact, incubation of Ishikawa cells with close-to-physiological concentrations of estradiol (E₂) as precursor resulted in: (1) elevated formation (up to 90%) of E₂-sulphate (E₂-S), using lower precursor concentrations; (2) very limited conversion to estrone (E₁) (< 10% at 24 h incubation), as either free or sulphate; and (3) low but consistent production of other estrogen derivatives, such as 2-hydroxy-estrogens and estriol. Conversely, scant amounts (if any) of E₂-S were found in HEC-1A cells, while no detectable formation of other estrogen metabolites could be observed after 24 h. On the other hand, E₁ production was significantly greater (nearly 60% at 24 h) than in Ishikawa cells, a large proportion of E₁ (over 50% of the total) being formed after only 6 h incubation using time-course experiments. The hypothesis that E₂ metabolism could be minor in Ishikawa cells as a consequence of the high rate of E₂-S formation encountered is contradicted by the evidence that conversion to E₁ also remains limited in the presence of much lower E₂-S amounts, seen using higher molar concentrations of precursor. Overall, we observe that 17β-hydroxysteroid dehydrogenase (17β-HSD) activity diverges significantly in intact Ishikawa and HEC-1A endometrial cancer cells. This difference could not merely be accounted for by the diverse amounts of substrate (E₂) available to the cells, nor may it be imputed to different levels of endogenous estrogens. It should rather be sought in different mechanisms controlling 17β-HSD activity or, alternatively, in the presence of distinct isoenzymes in the two different cell types.     

Oestradiol synthesis from oestrone in malignant breast epithelial cells: Studies on a high affinity, 80 kDa form of oestradiol dehydrogenase

Previous studies have shown that in the breast there are multiple forms of the enzyme oestradiol dehydrogenase (E₂DH), responsible for the interconversion of oestrone (E₁) to oestradiol (E₂). We have now re-examined oestrogen metabolism in the breast cancer cell lines (T47D and MCF-7) and have shown that steroids previously shown to inhibit the conversion of E₁ to E₂ in normal breast tissue failed to do so when added to growing monolayers of these malignant cells. In contrast to earlier estimates in normal breast tissues, the apparent K_m for this conversion in monolayers of these malignant cells is shown here to be considerably lower, at around 50 nM. Cell free studies on these cell lines have revealed the presence of a high affinity (for E₁) form of this enzyme of M_W 80 kDa. The ability to detect this enzyme in soluble cell fractions appears to be critically dependent on buffer composition. Normal breast epithelial cells and adipose tissue appear to be devoid of this form of E₂DH. As this form of E₂DH has the highest affinity for the substrate E₁ of all the forms in the breast, it is probable that this 80 kDa enzyme is responsible for the conversion of E₁ to E₂ in cell monolayers. If the observation holds that the 80 kDa enzyme is absent in the normal tissues, then the possibility arises that this E₂DH may be linked with the neoplastic process in some breast tumours containing malignant epithelial cells of a similar type as studied here.     

Emerging principles for the development of resistance to antihormonal therapy: implications for the clinical utility of fulvestrant

We seek to evaluate the clinical consequences of resistance to antihormonal therapy by studying analogous animal xenograft models. Two approaches were taken: (1) MCF-7 tumors were serially transplanted into selective estrogen receptor modulator (SERM)-treated immunocompromised mice to mimic 5 years of SERM treatment. The studies in vivo were designed to replicate the development of acquired resistance to SERMs over years of clinical exposure. (2) MCF-7 cells were cultured long-term under SERM-treated or estrogen withdrawn conditions (to mimic aromatase inhibitors), and then injected into mice to generate endocrine-resistant xenografts. These tumor models have allowed us to define Phase I and Phase II antihormonal resistance according to their responses to E₂ and fulvestrant. Phase I SERM-resistant tumors were growth stimulated in response to estradiol (E₂), but paradoxically, Phase II SERM and estrogen withdrawn-resistant tumors were growth inhibited by E₂. Fulvestrant did not support growth of Phases I and II SERM-resistant tumors, but did allow growth of Phase II estrogen withdrawn-resistant tumors. Importantly, fulvestrant plus E₂ in Phase II antihormone-resistant tumors reversed the E₂-induced inhibition and instead resulted in growth stimulation. These data have important clinical implications. Based on these and prior laboratory findings, we propose a clinical strategy for optimal third-line therapy: patients who have responded to and then failed at least two antihormonal treatments may respond favorably to short-term low-dose estrogen due to E₂-induced apoptosis, followed by treatment with fulvestrant plus an aromatase inhibitor to maintain low tumor burden and avoid a negative interaction between physiologic E₂ and fulvestrant.     

Distribution of 17β-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues

The interconversion of estrone (E₁) and 17β-estradiol (E₂), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3β,17β-diol is catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD). The enzyme 17β-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17β-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17β-HSD mRNA in some human tissues. Enzymatic activity was measured using ¹⁴C-labeled E₁, E₂, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highestlevel of 17β-HSD activity, low but significant levels of E₂ as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E₂→E₁, T→4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17β-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.     

Some observations on the measurement of estradiol-17β in human plasma by radioimmunoassay

The use of specific and non-specific antisera for estradiol-17β (E₂17β) were compared in the radioimmunoassay of the steroid. The effects of various “blank” mateirials on the standard curve and on the accuracy of recovery of E₂17β added to plasma before and after chromatography on LH-20 Sephadex were examined. It was concluded that the use of the specific antiserum (anti-6-oxoE₂17β -6-(O-carboxymethyl)oxime-bovine serum albumin(antiE₂17β-6-BSA) was an improvement on the non-specific serum anti-E₂17β-17-hemisuccinyl-bovine serum albumin (antiE₂ 17β-17-BSA) following chromatography of extracts. However, although a precise result could be obtained with the anti-E₂17β-6-BSA without the Chromatographic step, recovery of E₂17β added to plasma was only possible if the step was included.

The cross-reactivity of estrone (E₁)with E₂17β using anti-E₂17β-17-BSA as defined by Abraham (J. Clin. Endocr. , 866 (1969) was examined under conditions of constant and of changing E₁:E₂17β ratio.     

Estrogen stimulation of creatine kinase B specific activity in 3T3L1 adipocytes after their differentiation in culture: Dependence on estrogen receptor

We have demonstrated previously that rat adipose tissue showed sex and depot-specific responses to gonadal steroids. The epididymal fat pad in males responded exclusively to androgens by increased specific activity of the brain type isozyme of creatine kinase (CK). In females, the parametrial adipose tissue responded exclusively to estrogens. The present study was undertaken to follow the responsiveness to steroid hormones, and the presence of estrogen receptors (ER), in 3T3L1 cells during their differentiation from pre-adipocytes to adipocytes. In pre-adipocytes in which the basal CK specific activity is low, there was no CK response to 17β estradiol (E₂) or dihydrotestosterone (DHT). Differentiation of the cells into adipocytes was accompanied by increased basal CK activity which was stimulated by E₂, but not by DHT. Responsiveness to E₂ began 5 days after switching pre-adipocytes to differentiation medium. Upon differentiation, ER became demonstrable in the cell nuclei by staining with FITC labeled anti-idiotypic antibody (clone 1D5) directed against the steroid binding domain of ER. The response to E₂ was time-dependent and blocked completely by cycloheximide or actinomycin D. 1D5 itself, which has an estrogen mimetic effect, stimulated CK activity in the cells similarly to E₂. The antiestrogen tamoxifen which also stimulated CK activity in the adipocytes, completely blocked E₂ action. The ‘pure’ antagonist of E₂, ICI 164,384 and the tissue-selective antiestrogens, raloxifene or tamoxifen methiodide were also complete antagonists with no agonistic effects. The response of the 3T3L1 adipocytes to E₂ was upregulated by 1,25(OH)₂D₃. Moreover, IGF1 was also a potent stimulator of CK in these cells, and therefore may mediate partially the stimulation by E₂. Transient transfection of the pre-adipocytes with ER permitted E₂ induction of CK. Thus, the appearance of ER and concomitant responsiveness to E₂ is another hormone-related change occurring in 3T3L1 cells during differentiation, in addition to changes such as development of insulin responsiveness. The interactions in this system provide a useful in vitro model for investigating the development of responsiveness to E₂.     

Effect of prostaglandins on protein, RNA, DNA and collagen synthesis in experimental wounds

The effect of exogenous prostaglandins E₁, E₂ and F₂ (PGE₁, PGE₂ and PGF₂) on ³H-leucine, ³H-uridine, ³H-thymidine and ³H-proline incorporation in experimental cutaneous wounds has been studied in rats.

Prostaglandins E₁ and E₂ markedly stimulate the incorporation of these tritiated precursors, into protein, RNA, DNA and collagen synthesis, whereas F₂ inhibits it. All tested prostaglandins exhibit their maximum effect within the first hours following administration. Most active is PGE₁. These observations indicate that application of prostaglandins significantly stimulate incorporation with protein, RNA, DNA and collagen synthesis in the skin of wounded rats and thus, may play a role in epidermal cell growth and division as well as in scar-forming tissue.