Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Peripheral oxytocin treatment modulates central dopamine transmission in the mouse limbic structures

The effects of subcutaneous (s.c.) oxytocin treatment have been investigated on various parameters of dopaminergic neurotransmission in basal forebrain structures (nucleus olfactorius posterior + nucleus accumbens + septum) of the mouse. Acute oxytocin treatment failed to influence dopamine utilization in the basal forebrain. Following chronic injections of oxytocin (0.2 mg/kg) for 8 8 days, the neuropeptide decreased dopamine utilization. Neither in vivo nor in vitro oxytocin treatment was capable of influencing the in vitro uptake of [³H]dopamine in basal forebrain slices. The spontaneous release of [³H]dopamine (in the presence of 4.2 mM K⁺) from basal forebrain tissue slices was not affected by in vitro or acute or chronic in vivo oxytocin treatment. The stimulated release of [³H]dopamine (in the presence of 30 mM K⁺) was significantly inhibited by chronic in vivo oxytocin administration. Chronic oxytocin treatment decreased the B_max value of [³H]spiroperidol binding in the basal forebrain. The dissociation constant (K_d) of [³H]spiroperidol binding was not influenced by oxytocin. The data indicate that peripheral oxytocin treatment is capable of modifying dopaminergic neurotransmission in mouse basal forebrain regions.     

Flavonoids protect against oxidative damage to LDL in vitro: use in selection of a flavonoid rich diet and relevance to LDL oxidation resistance ex vivo?

The ability of a range of dietary flavonoids to inhibit low-density lipoprotein (LDL) oxidation in vitro was tested using a number of different methods to assess oxidative damage to LDL. Overall quercetin was the most effective inhibitor of oxidative damage to LDL in vitro. On this basis, a diet enriched with onions and black tea was selected for a dietary intervention study that compared the effect on the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo in healthy human subjects of a high flavonoid diet compared with a low flavonoid diet. No significant difference was found in the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo between the high flavonoid and low flavonoid dietary treatments (48 ± 1.6 min compared to 49 ± 2.1 min).     

肺癌患者外周血中循环肿瘤细胞的快速分离与体外培养

循环肿瘤细胞(circulating tumor cells, CTCs)是从肿瘤病灶脱落并进入外周血液循环的处于游离状态的肿瘤细胞,代表了肿瘤病灶的分子特征,可用于对肿瘤的“液体活检”。但外周血中CTCs数目极为稀少,使得后续针对CTCs的分子与功能分析面临巨大挑战。鉴于此,本文建立了一种基于微流控芯片和免疫磁珠的能够快速从肺癌患者的外周血中分离CTCs的方法。该方法直接针对全血进行一步分离,可避免血液样本预处理及富集等过程对细胞造成的损伤,从而有效地保护CTCs的活性(>90%)。分离得到的CTCs可富集在小体积中(80 μL),实现高密度的细胞培养,完成体外扩增,扩增后的CTCs可以被进一步冻存、复苏及再次增殖培养,表明已经对患者血液中的CTCs成功建系。本文进一步对CTCs进行了基因突变(EGFR、KRAS、PIK3CA、TP53和BRAF)检测及荧光标记葡萄糖类似物(2-NBDG)摄取的功能分析,证明CTCs存在较大异质性。本研究成功实现了对外周血中稀少的CTCs进行体外培养,并对CTCs进行了基因、蛋白、功能等各个层面的分析,这对于肿瘤精准医疗具有重要的临床意义。     

Monosialoganglioside increases catalase activity in cerebral cortex of rats

Monosialoganglioside (GM1) is a neuroprotective agent that has been reported to scavenge free radicals generated during reperfusion and to protect receptors and enzymes from oxidative damage. However, only a few studies have attempted to investigate the effects of GM1 on enzymatic antioxidant defenses of the brain. In the present study, we evaluate the effects of the systemic administration of GM1 on the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and on spontaneous chemiluminescence and total radical-trapping potential (TRAP) in cerebral cortex of rats ex vivo. The effects of GM1 on CAT activity and spontaneous chemiluminescence in vitro were also determined.

Animals received two injections of GM1 (50 mg/kg, i.p.) or saline (0.85% NaCl, i.p.) spaced 24 h apart. Thirty minutes after the second injection the animals were sacrificed and enzyme activities and spontaneous chemiluminescence and TRAP were measured in cell-free homogenates. GM1 administration reduced spontaneous chemiluminescence and increased catalase activity ex vivo, but had no effect on TRAP, SOD or GSH-Px activities. GM1, at high concentrations, reduced CAT activity in vitro. We suggest that the antioxidant activity of GM1 ganglioside in the cerebral cortex may be due to an increased catalase activity.     

In vitro steroid metabolic studies in human testes II: Metabolism of cholesterol, pregnenolone, progesterone, androstenedione and testosterone by testes of an estrogen-treated man

The effect of long-term in vivo estrogen treatment on in vitro steroidogenesis by the testes of a young man was investigated. In vitro incubation of testicular tissue of this man with ³H-pregnenolone, ³H-progesterone, ³H-androstenedione and ³H-testosterone demonstrated suppression of 17-hydroxylase activity, with little or no effect of the treatment on Δ⁵-3β-hydroxysteroid oxidoreductase, 5a-reductase and aromatase. Increased 20-hydroxysteroid oxidoreductase activity was observed. Determination of intratesticular steroid concentrations led to similar conclusions.     

Purine enzyme inhibition fails to alter benzodiazepine receptor binding in brain

Binding of [³H]flunitrazepam to benzodiazepine receptors in brain from several species, including human, was measured in vitro in the presence and absence of purine-metabolizing enzyme inhibitors. Incubation with potent inhibitors of either adenosine deaminase (2′-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)-adenine) or guanine deaminase (5-amino-4-imidazole carboxamide) failed to alter [³H]flunitrazepam binding in homogenates of several different regions of human, rabbit, rat or guinea pig brain. These findings are in contrast to those of Norstrand et al. [Enzyme 29, 61–65 (1983)] who reported substantial alterations in [³H]flunitrazepam binding to human brain membranes in the presence of erythro-9-(2-hydroxy-3-nonyl)-adenine (increase) and 5-amino-4-imidazole carboxamide (decrease). In our studies, [³H]flunitrazepam binding was also unaltered in more anatomically intact brain sections following treatment with purine enzyme inhibitors. Furthermore, in vivo administration of erythro-9-(2-hydroxy-3-nonyl)-adenine to mice at a dose (200 mg/kg, i.p.) known to almost totally inhibit central adenosine deaminase activity also failed to alter brain [³H]flunitrazepam binding measured ex vivo, 30–120 min post injection.

While previous studies have shown that purines such as inosine interact with benzodiazepine receptors, our results raise some questions about the role of endogenous purines in regulating benzodiazepine receptors, at least in vitro and also acutely _vivo following purine enzyme inhibitor administration.     

Tissue selective action of tamoxifen methiodide, raloxifene and tamoxifen on creatine kinase B activity in vitro and in vivo

We have compared the cell and tissue selective estrogenic and antiestrogenic activities of tamoxifen, raloxifene, ICI 164,384 and a permanently ionized derivative of tamoxifen — tamoxifen methiodide (TMI). This non-steroidal antiestrogen has limited ability to cross the blood brain barrier and is therefore less likely to cause the central nervous system disturbances caused by tamoxifen. We have used the stimulation of the specific activity of the “estrogen induced protein”, creatine kinase BB, as a response marker in bone, cartilage, uterine and adipose cells and in rat skeletal tissues, uterus and mesometrial adipose tissue. In vitro, TMI, tamoxifen and raloxifene mimicked the agonistic action of 17β-estradiol in ROS 17/2.8 rat osteogenic osteosarcoma, female calvaria, and SaOS₂ human osteoblast cells. In Ishikawa endometrial cancer cells, tamoxifen showed reduced agonistic effects and raloxifene showed no stimulation. However, as antagonists, tamoxifen and raloxifene were equally effective in Ishikawa or SaOS₂ cells. In immature rats, all four of the antiestrogens inhibited estrogen action in diaphysis, epiphysis, uterus and mesometrial adipose tissue; when administered alone, tamoxifen stimulated creatine kinase (CK) specific activity in all these tissues. Raloxifene and TMI, however, stimulated only the skeletal tissues and had no stimulatory effect in the uterus or mesometrial fat, and the pure antiestrogen ICI 164,384 showed no stimulatory effect in any of the tissues. The simultaneous injection of estrogen, plus an antiestrogen which acted as an agonist, resulted in lower CK activity than after injection of either agent alone. These differential effects, in vivo and in vitro, may point the way to a wider therapeutic choice of an appropriate antiestrogen which, although antagonizing E₂ action in mammary cancer, can still protect against osteoporosis and cardiovascular disease and not stimulate the uterus with its attendant undesirable changes, or interfere with the beneficial action of E₂ in the brain.     

Biochemical markers in preclinical models of osteoporosis

Although several treatments for osteoporosis exist, further understanding of the mode of action of current treatments, as well as development of novel treatments, are of interest. Thus, preclinical models of osteoporosis are very useful, as they provide the possibility for gaining knowledge about the cellular mechanisms underlying the disease and for studying pharmaceutical prevention or intervention of the disease in simple and strictly controlled systems. In this review, we present a comprehensive collection of studies using biochemical markers of bone turnover for investigation of preclinical models of osteoporosis. These range from pure and simple in vitro systems, such as osteoclast cultures, to ex vivo models, such as cultures of embryonic murine tibiae and, finally, to in vivo models, such as ovariectomy and orchidectomy of rats. We discuss the relevance of the markers in the individual models, and compare their responses to those observed using 'golden standard' methods.     

Responsiveness of rat ovaries to dcAMP in the perinatal period: evidence for an inhibitory influence in vivo.

The progesterone production by rat ovaries from 18-day-old fetuses to 6-day-old neonates was measured in vitro in the presence of dibutyryl cAMP (dcAMP, 1 mM). A pronounced decline was observed at the end of fetal life. The 5-reductase activity did not seem sufficient to explain this decrease. Preculture of the ovaries for 48 h in the basal medium enhanced responsiveness to the nucleotide. Addition of spironolactone, an inhibitor of 17-hydroxylase to dcAMP did not modify this evolution.

3β-hydroxysteroid dehydrogenase activity, detectable in fetal ovaries in the absence of dcAMP was also increased after preculture. In the presence of spironolactone and trilostane, the pregnenolone production showed the same evolution as progesterone and was also enhanced after culture. These results suggest the existence of inhibitory factor(s) present in vivo at the end of fetal life.     

Regulation of proliferation of rat cartilage and bone by sex steroid hormones

We have demonstrated previously that 17β-estradiol (E₂) stimulates proliferation of skeletal tissues, both in vivo and in vitro, as measured by increased DNA synthesis and creatine kinase (CK) specific activity. The effect of E₂ on bone is sex specific. E₂ is active only in females and androgens only in males. By contrast, in cartilage of both sexes, dihydrotestosterone (DHT) as well as E₂ stimulates CK specific activity and DNA synthesis. In bone, we find that sex steroids stimulate skeletal cell proliferation in gonadectomized as well as in immature rats. Ovariectomized (OVX) rats, between 1 and 4 weeks after surgery, show stimulation of CK by E₂. The basal activity and response of CK changes with the varying endogenous levels of E₂ in cycling rats, in which the highest basal activity is at proestrus and estrus and the highest response is in diestrus. In rats of all ages tested, both the basal and stimulated specific activity of CK is higher in diaphysis and epiphysis than in the uterus, or in the adipose tissue adjacent to the uterus, which has a response similar to that of the uterus itself. The effect of E₂ in vivo, and in chrondroblasts and osteoblasts in vitro, is inhibited by high levels of the antiestrogen tamoxifen which, by itself, in similar high concentrations, shows stimulatory effects. In addition to the sex steroids, skeletal cells are also stimulated by secosteroid and peptide calciotrophic hormones. The interactions of the sex steroids and the other calciotrophic hormones. These results provide the first steps towards understanding the regulation of bone cell proliferation and growth by the concerted action of a variety of hormones and growth factors.     

Phenolic A ring requirement for the neuroprotective effects of steroids

Estrogens are reported to reduce the incidence of Alzheimer's disease and 17β-estradiol (βE2), the potent, naturally occurring estrogen, exerts neuroprotective effects in a variety of in vivo and in vitro model systems. The present study elucidates the structural requirements of steroids and related compounds for neuroprotectivity at low nM doses. All estrogens tested with an intact phenolic A ring protected SK---N---SH neuroblastoma cells from the toxic effects of serum-deprivation. All 3-O-methyl ether cogeners tested were inactive indicating the importance of a phenolic A ring. The diphenolic estrogen mimic diethylstilbesterol (DES) was neuroprotective and retention of a single phenolic function was sufficient to retain neuroprotective activity. The di-O-methyl ether of DES was inactive. The following steroids which lack a phenolic A ring were also inactive: testosterone; dihydrotestosterone; progesterone; corticosterone; prednisolone; 6 -methylprednisolone; aldosterone; and cholesterol. Finally, phenol, lipophilic phenols, and tetrahydronapthol were inactive. These results suggest that a phenolic A ring and at least three rings of the steroid nucleus are necessary for the neuroprotective activity of estrogens.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

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.     

Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production

Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction. With the increasing demand for transfusion, however, there is an undersupply of donors to provide the platelet source. Thus, scientists have sought to design methods for deriving clinical-scale platelets ex vivo. Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells (SCs), the platelet yields achieved using these strategies have not been adequate for clinical application. In this review, we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo, recapitulate the key advances in the production of SC-derived platelets using several SC sources, and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.     

The metabolism of dihydrotachysterols: Renal side chain and non-renal nuclear hydroxylations in vivo and in vitro

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT₂ and DHT₃. Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT₂) was demonstrated in human plasma after administration of oral DHT₂. Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT₃ in vitro showed that 25-hydroxy-DHT₃ was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT₃-23,26-lactone and 23,25-dihydroxy-24-oxo-DHT₃. The osteosarcoma cell line metabolized 25-OH-DHT₃ in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1,25-dihydroxy-DHT₃. The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1,25-dihydroxy-DHT whereas compound Hb is possibly 1β,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1,25-dihydroxy-DHT is very effective in displacing radiolabelled 1,25-dihydroxyvitamin-D₃ and is thus most likely to be the calcaemic metabolite of DHT.     

Studies on the immune response of sheep to Chabertia ovina (Fabricius, 1788)

The immunological basis of host resistance to C. ovina was established by the demonstration of a marked anamnestic response by 6 sheep to a second infection of C. ovina after previous sensitization. Reaginic antibody was detected in the circulation of 20 sheep tested during both primary and secondary infection. It was first detected 5 weeks after infection with 2000 larvae, 8 weeks after infection with 1000 larvae and 12 weeks after infection with 400 larvae. In contrast, precipitating antibody was detected in only 10 per cent of sheep tested, while in vivo and in vitro tests for delayed hypersensitivity were negative. Studies on the effect of antilymphocytic serum on immunity to C. ovina suggested that the development of resistance to C. ovina requires the participation of the thymus-derived lymphocyte.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Specificity of the action of parathyroid hormone upon mitochondrial metabolism: Cyanogen bromide-derived parathyroid-hormone peptides

The mitochondrial response to cyanogen bromide-treated parathyroid hormone was studied as a means of testing further the relationship between the structure and the effects in vitro of this hormone. The treated hormone and appropriate control hormone were tested in a standard bioassay and in a mitochondrial assay system in vitro.

Reaction of more than 90 % of the methionine residues in the hormone resulted in total inactivation of the hormone both in vivo and in vitro. This result disagrees with previously published data.