Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

Cultured hepatoma cells for the study of enzyme regulation: Induction of ornithine decarboxylase by insulin and asparagine

Summary The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combintion, of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10⁻⁵ M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present. This work was supported in part by Grants CA-07175, CA-22484, and CA-17334 from the National Cancer Institute. D. P. G. is a Predoctoral Fellow at the Food Research Institute, supported by a fellowship from the Monsanto Fund and by NIH Grant R01-AI 15693 to Prof. Michael W. Pariza, Food Research Institute, University of Wisconsin, Madison.     

Effects of antiestrogen versus antiprogestin on transformed and nontransformed steroid receptors

In order to determine if different physicochemical properties exist among antihormone-receptor complexes, we have compared the interaction of the antiprogestin RU486 with progesterone receptor (PR) versus the triphenylethylene antiestrogen H1285 (4-(N,N-diethyl-aminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl-alp ha'- ethylstilbene] with estrogen receptor (ER) from rabbit uterine tissue. Contrary to other reports, we observed no difference in the sedimentation properties of transformed PR (4S) when bound by the antagonist RU486 versus the progesterone agonist R5020 in either cytosol or DEAE partially-purified receptor preparations analyzed on sucrose gradients containing 0.3 M KCl. In addition, we found no difference in the sedimentation properties of these receptor preparations in the presence of 10 mM sodium molybdate: the nontransformed RU486-PR and nontransformed R5020-PR both sedimented as a 6S species. These same results were obtained when the receptor preparation and gradient analysis were performed in the absence of monothioglycerol. Likewise, there was no change in the sedimentation properties of the transformed PR when the receptor, partially purified in the absence of molybdate, was analyzed on sucrose gradients containing 10 mM sodium molybdate to prevent receptor alteration during centrifugation. From DNA-cellulose assays performed with partially purified PR in the absence of molybdate we determined that the 4S form of R5020-PR and RU486-PR is transformed receptor; whereas in the presence of molybdate, the 6S species is nontransformed. In contrast, we found a different pattern of sedimentation when comparing transformed antiestrogen-receptor complexes with transformed estrogen-receptor complexes. In this case, transformed H1285-ER sedimented as 6S and estradiol-ER sedimented as 4S. We conclude from these experiments that these two antihormones, RU486 and H1285, may have different mechanisms of action in their antagonism of steroid hormone action. Antiestrogen stabilizes the salt-transformed ER as a dimer while antiprogestin appears to permit dissociation of the oligomeric form of the receptor to the monomeric form.     

Antiestrogen action: Differential nuclear retention and extractability of the estrogen receptor

Since there is a much longer uterine nuclear retention of the U-11,100A (antiestrogen) receptor complex (UARC) than of the estradiol receptor complex (ERC) at 4–12 hrs after injection, experiments were designed to determine if there is a difference between the relative nuclear affinities for the two RCs as determined by extraction with various ionic strength mediums. Although the UARC was retained longer in the nuclear fraction $\text{in}$$\text{vivo}$, the UARC was completely extractable with 0.3M KC1 or 50mM spermine, whereas the ERC demonstrates a saltresistant form. This suggests that the ERC is more tightly bound to nuclear components through this salt-resistant form of the receptor. In addition, various intercalating agents were used to distinguish the different nuclear chromatin DNA sites where the UARC and ERC may be binding. With actinomycin D (50 uM) more ERC than UARC was retained in the nuclear fraction. However, with ethidium bromide (100uM) less ERC than UARC was retained. Also, the ERC selectively released by ethidium bromide is precisely that fraction not released by salt. These results indicate that the UARC and ERC bind to different chromatin loci.     

Ala54Thr Fatty Acid-Binding Protein 2 (FABP2) Polymorphism in Recurrent Depression: Associations with Fatty Acid Concentrations and Waist Circumference

Background

Fatty acid (FA)-alterations may mediate the mutual association between Major Depressive Disorder (MDD) and cardiovascular disease (CVD). However, etiology of observed FA-alterations in MDD and CVD remains largely unclear. An interesting candidate may be a mutation in the fatty acid–binding protein 2 (FABP2)-gene, because it regulates dietary FA-uptake. Therefore, we aimed to test the hypotheses that in MDD-patients the FABP2 Ala54Thr-polymorphism would be (I) more prevalent than in sex- and age-matched controls, (II) associated with observed alterations in FA-metabolism, and (III) associated with CVD-risk factor waist circumference.

Methods

We measured concentrations of 29 different erythrocyte FAs, FABP2-genotype, and waist circumference in recurrent MDD-patients and matched never-depressed controls.

Results

FABP2-genotype distribution did not significantly differ between the 137 MDD-patients and 73 matched controls. However, patients with the Ala54Thr-polymorphism had (I) higher concentrations of especially eicosadienoic acid (C20:2ω6; P=.009) and other 20-carbon FAs, and associated (II) lower waist circumference (P=.019). In addition, FABP2-genotype effects on waist circumference in patients seemed (I) mediated by its effect on C20:2ω6, and (II) different from controls.

Conclusions

Although Ala54Thr-polymorphism distribution was not associated with recurrent MDD, our results indicate that FABP2 may play a role in the explanation of observed FA-alterations in MDD. For Ala54Thr-polymorphism patients, potentially adaptive conversion of increased bioavailable dietary precursors into eicosadienoic acid instead of arachidonic acid might be related to a low waist circumference. Because this is the first investigation of these associations, replication is warranted, preferably by nutrigenetic studies applying lipidomics and detailed dietary assessment.     

A power analysis for future clinical trials on the potential adverse effects of SSRIs on amygdala reactivity

Treatment of adolescents with antidepressants may induce an increased risk for suicidality in this population. The activity of the amygdala during processing of emotional faces with functional Magnetic Resonance Imaging (fMRI) is a well-known measure of emotional dysregulation. Based upon data of our prematurely ended randomized clinical trial with fluoxetine (NTR3103) in anxious and or depressed girls (12–14 years of age) we calculated that with the found effect size of r = 0.66, compared to placebo, only 8 subjects are needed to demonstrate increased amygdala activity following 16 weeks of treatment with fluoxetine.     

Environmental filtering determines community patterns in temporary wetlands: a multi-taxon approach

Climate characteristics appear to play a key role in filtering organisms based on their biological traits. If this trait filtering by climate indeed occurs, it should have effects on the composition, dynamics, taxonomic relatedness and co-occurrence patterns of local assemblages, regardless of the taxonomic group considered. This preliminary study aimed to assess the extent to which environmental variables might determine these patterns in local communities and to evaluate whether the ultimate cross-taxon congruence relationships are consistent across, or dependent on, the selected region. To this end, we studied the bryophyte, macrophyte, macroinvertebrate, and amphibian communities in two clusters of temporary wetlands on the NE Iberian Peninsula under mesothermal and semiarid climates. We observed effects of environmental filtering, with the communities differing between the climatic regions not only in their compositions but also in their dynamics and taxonomic relatedness patterns. Although the cross-taxon congruence in terms of species richness was high in the mesothermal climate, most of the congruent relationships were disrupted in the semiarid environment. Overall, because climate-dependent patterns appear to prevail over climate-consistent ones, we suggest that the use of surrogate taxa may be of limited value when aiming to assess wetland biodiversity across large areas.     

Ultraviolet photosensitivity of the estrogen binding protein from rat uterus. Wavelength and ligand dependence. Photocovalent attachment of estrogens to proteins.

Ultraviolet irradiation of the estrogen binding protein in rat uterine cytosol results in a progressive photoinactivation which is rapid at 254 nm and slower at greater than 315 nm. Both unfilled and estradiol-filled sites are inactivated at approimately the same rates at 254 nm (t 1/2 equals 8 min and 11 min, respectively), but at 315 nm, empty sites are consumed much more rapidly (t 1/2 equals 3.4 hr) than filled ones (t 1/2 equals 24 hr). The protective effect of the estrogen ligand at this wavelength appears to depend on its binding to the estrogen-specific binding site, as inactivation rate studies at different concentrations of estrone, estradiol, and estriol show a good correlation between the extent of protection and the fractional saturation of the high affinity estrogen binding sites. Scatchard analysis indicates that inactivation is the result of a loss of binding sites and not a decrease in their affinity, and sedimentation analysis shows that increased heterogeneity and aggregation of the estrogen binding species accompanies the photoinactivation process. Photoinactivation appears to be the result of direct irradiative damage of the animo acid residues, as the inactivation rate is the same under air and nitrogen atmospheres, and is unaffected by nucleophiles, reductants, and radical scavengers. When photoinactivation is measureed by irradiation of cytosol containing [3-H]estradiol, a concurrent photocovalent attachment process is noted; the steroid becomes linked to protein in a solvent-extractable manner (boiling ethanol inextractable). This attachment, however, does not appear to be related to the steroid binding at the estrogen binding site. Its rate is affected by reductants and scavengers. A similar photocovalent attachment reaction occurs when bovine serum albumin or ovalbumin is irradiated in the presence of [3-H]estradiol or [3-H]diethylstilbestrol. The detailed reactions involved in this photocovalent attachment process have not been defined at present.     

Flow regulation increases food‐chain length through omnivory mechanisms in a Mediterranean river network

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