Estrogen receptor-anti-estrogen complex: atypical binding by uterine nuclei and effects on uterine growth

A single injection of the anti-estrogen, nafoxidine hydrochloride (Upjohn 11,100 A), causes the retention of the estrogen receptor by uterine nuclei for as long as 19 days. This is accompanied by the long term stimulation of uterine growth. The nuclear retention of the receptor does not stimulate the replenishment of the cytoplasmic receptor and failure to do so is suggested as the mechanism by which nafoxidine acts as an anti-estrogen.     

The effects of protein synthesis inhibitors on oxidative phosphorylation by plant mitochondria

Inhibitors can be successfully used if they are specific for only one process. Published data suggest that some inhibitors of protein synthesis may also inhibit respiration or oxidative phosphorylation. The effect of a range of protein synthesis inhibitors on respiration and phosphorylation has been studied, using tightly coupled mitochondria from several plant species including turnips (Brassica napus).Puromycin, actinomycin D, lincomycin, mitomycin C and d-serine did not uncouple or inhibit respiration. Cycloheximide caused a partial inhibition (maximum 22% at 3 mM) of malate but not succinate-driven respiration. Chloramphenicol was a potent inhibitor of electron transport, but not of phosphorylation. The activity of the isomers of chloramphenicol varied in the order l-threo >d-threo >l-erythro >d-erythro. From evidence presented it is concluded that chloramphenicol has three sites of action, the flavoprotein level being most sensitive, the second site of variable sensitivity lies between cytochromes b and c and the third site at the cytochrome a level is only slightly affected by the inhibitor.     

Differential effects of protein synthesis inhibitors on porcine oocyte activation

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L-[³⁵S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D-SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25-kDa polypeptide to a 22-kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley-Liss, Inc.     

Estrogen and antiestrogen effects on neonatal ovine uterine development

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. These critical events coincide with expression of estrogen receptor alpha (ERalpha) by nascent endometrial glands and stroma. To test the working hypothesis that estrogen and uterine ERalpha regulate uterine growth and endometrial gland morphogenesis in the neonatal ewe, ewes were treated daily from birth (PND 0) to PND 55 with 1) saline and corn oil as a vehicle control (CX), 2) estradiol-17 beta (E2) valerate (EV), an ERalpha agonist, 3) EM-800, an ERalpha antagonist, or 4) CGS 20267, a nonsteroidal aromatase inhibitor. On PND 14, ewes were hemihysterectomized, and the ipsilateral oviduct and ovary were removed. The remaining uterine horn, oviduct, and ovary were removed on PND 56. Treatment with CGS 20267 decreased plasma E2 levels, whereas EM-800 had no effect compared with CX ewes. Uterine horn weight and length were not affected by EM-800 or CGS 20267 but were decreased in EV ewes on PND 56. On PND 14 and PND 56, treatment with EV decreased endometrial thickness but increased myometrial thickness. The numbers of ductal gland invaginations and endometrial glands were not affected by CGS but were lower in EM-800 ewes on PND 56. Exposure to EV completely inhibited endometrial gland development and induced luminal epithelial hypertrophy but did not alter uterine cell proliferation. Exposure to EV substantially decreased expression of ERalpha, insulin-like growth factor (IGF) I, and IGF-II in the endometrium. Results indicate that circulating E2 does not regulate endometrial gland differentiation or development. Although ERalpha does not regulate initial differentiation of the endometrial glandular epithelium, results indicate that ERalpha does regulate, in part, coiling and branching morphogenesis of endometrial glands in the neonatal ewe. Ablation of endometrial gland genesis by EV indicates that postnatal uterine development is extremely sensitive to the detrimental effects of inappropriate steroid exposure.     

Paradoxical effects of protein synthesis inhibitors on uridine uptake in cultured cells: Possible role of uncharged tRNA in regulating metabolism

Previous studies (J. Biol. Chem, 253: 99–105, 1978) showed that thyrotropin-releasing hormone (TRH) acutely stimulated uridine uptake in pituitary cell (GH₄C₁) cultures. Studies on the role of protein synthesis in this response to TRH led to the finding that an inhibitor of ribosomal translation, cycloheximide, also stimulated uridine uptake acutely. Studies reported here attempt to determine the mechanism of cycloheximide action and whether cycloheximide and hormone stimulation of uridine uptake occurred by similar pathways. The experiments presented indicate that: (1) seven inhibitors of ribosomal translation stimulated uridine uptake; (2) in contrast, inhibition of protein synthesis at tRNA aminoacylation resulted in reduced rates of uridine uptake; (3) inhibition of tRNA aminoacylation blocked cycloheximide but not TRH stimulation of uptake; (4) cycloheximide stimulation of uptake was restricted to amino acid-depleted cultures; (5) amino acid supplementation stimulated uridine uptake with a time-course identical to that of cycloheximide; (6) cycloheximide and amino acid supplementation promoted reacylation of cellular tRNAs in amino acid-depleted cultures; and (7) cycloheximide stimulation of uridine uptake resulted from enhanced nucleoside phosphorylation rather than increased uridine transport. We conclude that cycloheximide and amino acid stimulation of uridine phosphorylation may be mediated through a common pathway involving the extent of amino-acylation of cellular tRNAs. Furthermore, cycloheximide and TRH stimulate uridine phosphorylation by pathways that are distinguishable. It is apparent that not all cellular effects of cycloheximde can be attributed solely to inhibition of the synthesis of proteins.     

Differing effects of rapamycin and mTOR kinase inhibitors on protein synthesis

mTOR (mammalian target of rapamycin) forms two distinct types of complex, mTORC (mTOR complex) 1 and 2. Rapamycin inhibits some of the functions of mTORC1, whereas newly developed mTOR kinase inhibitors interfere with the actions of both types of complex. We have explored the effects of rapamycin and mTOR kinase inhibitors on general protein synthesis and, using a new stable isotope-labelling method, the synthesis of specific proteins. In HeLa cells, rapamycin only had a modest effect on total protein synthesis, whereas mTOR kinase inhibitors decreased protein synthesis by approx. 30%. This does not seem to be due to the ability of mTOR kinase inhibitors to block the binding of eIFs (eukaryotic initiation factors) eIF4G and eIF4E. Analysis of the effects of the inhibitors on the synthesis of specific proteins showed a spectrum of behaviours. As expected, synthesis of proteins encoded by mRNAs that contain a 5'-TOP (5'-terminal oligopyrimidine tract) was impaired by rapamycin, but more strongly by mTOR kinase inhibition. Several proteins not known to be encoded by 5'-TOP mRNAs also showed similar behaviour. Synthesis of proteins encoded by 'non-TOP' mRNAs was less inhibited by mTOR kinase inhibitors and especially by rapamycin. The implications of our findings are discussed.     

The effects of protein synthesis inhibitors on theGonyaulax clock

Summary Cycloheximide has been shown to be potent in phase-shifting the circadian glow rhythm of the dinoflagellateGonyaulax polyedra. In experiments in which the cells were exposed to drug pulses of varying concentration (0.35 M to 10 M) and duration (0.5 h to 8 h), the phase shift produced was linear with the log of the product of pulse strength and duration. The sensitivity to drug-induced phase shifting varies as a function of time of day; both advances and delays occurred and, depending on the strength of the perturbation, resulted in strong or weak-type phase response curves. Pulses given at different times after the light/dark to constant dim transition resulted in a crossover from delays to advances at about 15.5 h; this crossover point was the same at 19 °C and 24°C. The occurrence of extended transients following cycloheximide-induced phase advances (but not delays) appears to be the first example of such transients in a microbial circadian system.This research was supported in part by a grant from the National Institutes of Health (GM-19536). J.D. was a NIH predoctoral trainee (T01-GM 00036 and NRSA T32-GM 07598)     

Effect of estradiol on neuronal Swedish-mutated beta-amyloid precursor protein metabolism: reversal by astrocytic cells

Alzheimer's disease is the most frequent neurodegenerative disorder in the aged population and is characterized by the deposition of the 40/42-residue amyloid beta protein (Abeta), a proteolytic fragment of the beta-amyloid precursor protein (APP). Recently, it has been shown that physiological doses of estradiol reduce the generation of endogenous Abeta in primary cortical neurons. Here we investigate the influence of estrogen in amyloidogenesis and sAPPalpha secretion in the CNS. By means of primary cortical neurons overexpressing humanized APP(695) bearing the Swedish mutation (hAPP(695sw)), we analyzed APP maturation in the absence or in the presence of estrogen. We show that estrogen at a 2 microM concentration increases the release of the neuroprotective sAPPalpha fragment but does not reduce the release of Abeta in primary neurons overexpressing the Swedish-mutated form of APP. Furthermore, neurons cocultured with astrocytic cells or grown with astrocytes conditioned media do not exhibit the estrogen-induced increase in sAPPalpha secretion. Altogether, our data indicate that astrocytes interfere with estrogen in the regulation of sAPPalpha secretion, probably via secreted factor(s).     

Carbon source regulation of cephem antibiotic production by resting cells of Streptomyces clavuligerus and its reversal by protein synthesis inhibitors

The effect of utilizable carbon sources on the production of cephem antibiotics by Streptomyces clavuligerus has been studied. The pattern of utilizable carbon sources was found to be very restricted. Glycerol, maltose and starch supported the most extensive growth. Increasing the initial concentrations of carbon sources decreased both the volumetric and the specific production of cephems. A resting cell system was adopted for this study. Linear production of cephems continued for 4–7 h. The production rate of the resting cell system was higher with mycelia harvested at an early exponential stage than with those harvested at a late stage of growth. Addition of carbon source to the resting cell system decreased the production rate of cephems. This suppressive effect was prevented by the addition of chloramphenicol (or streptomycin) although uptake of carbon source was not inhibited by such a protein synthesis inhibitor.     

雌激素对淀粉样β蛋白代谢的调节和毒性缓解

以淀粉样β蛋白为主的老年斑胞外沉积和神经元内神经原纤维缠结,是阿尔采末病(AD)特征性的病理学改变。近来,人们逐渐认可淀粉样蛋白假说,即认为淀粉样蛋白沉积是AD最初起因。研究人员正在寻找针对淀粉样β蛋白沉积的药物,雌激素是其中之一。初步的工作证明,雌激素能够调节淀粉样β蛋白前体代谢,减少淀粉样β蛋白生成,也能够减轻淀粉样B蛋白引起的免疫炎症反应、氧应激对细胞造成的损伤,和对抗细胞凋亡。     

The effects of cosolutes on protein dynamics: the reversal of denaturant-induced protein fluctuations by trimethylamine N-oxide

The protein stabilizing effects of the small molecule osmolyte, trimethylamine N-oxide, against chemical denaturant was investigated by NMR spin-relaxation measurements and model-free analysis. In the presence of 0.7 M guanidine hydrochloride increased picosecond-nanosecond dynamics are observed in the protein ribonuclease A. These increased fluctuations occur throughout the protein, but the most significant increases in flexibility occur at positions believed to be the first to unfold. Addition of 0.35 M trimethylamine N-oxide to this destabilized form of ribonuclease results in significant rigidification of the protein backbone as assessed by (1)H-(15)N order parameters. Statistically, these order parameters are the same as those measured in native ribonuclease indicating that TMAO reduces the amplitude of backbone fluctuations in a destabilized protein. These data suggest that TMAO restricts the bond vector motions on the protein energy landscape to resemble those motions that occur in the native protein and points to a relation between stability and dynamics in this enzyme.