Mitochondrial redox state as a potential detector of liver dysoxia in vivo

Cyclin-dependent kinase 4 (Cdk4) activity is misregulated in most cancers. Loss of Cdk4 regulation can occur through overexpression of Cdk4 catalytic subunit or its regulatory partner cyclin D1, or if the Cdk4-specific inhibitory protein p16(INK4A) is inactive. We have attempted to express the two human subunits, Cdk4 and cyclin D1, in the yeast Saccharomyces cerevisiae. Surprisingly, expression of Cdk4 alone, under control of the strong GAL promoter, inhibits cell growth. Coexpression of both subunits allows formation of an active Cdk4-cyclin D1 complex which accentuates growth arrest. In cells expressing Cdk4 only, growth is restored by overexpressing human Cdc37, a Cdk4-binding molecular chaperone. Interestingly, the effect of Cdk4 on yeast is also overcome by both p16- and p21-families of Cdk-inhibitory proteins. Moreover, flavopiridol, a compound which inhibits Cdk4 enzyme activity, restores cell division. The fact that p16(INK4A) and flavopiridol negate Cdk4-mediated suppression of yeast cell growth implies that this simple system can be used as a screen for identifying Cdk4-specific antagonists which may mimic p16(INK4A) in the cancer cell cycle.     

Diversity beliefs as a mediator to faculty attitudes toward students with disabilities.

A survey of a sample of faculty (N = 201) at a large, public university located in the Southwest was conducted to investigate whether differences in faculty attitudes toward diversity positively mediate faculty attitudes toward persons with disabilities. In addition, the current study examined whether differences in faculty attitudes toward diversity may be viewed as positively mediating the relationship between instructor characteristics and their attitudes toward persons with disabilities. This study concludes that faculty members may not be viewing disability as part of the greater construct of diversity with empirical evidence. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Analysis of estrogen receptor interaction with tertiary-structured estrogen responsive elements

An initial crucial step in estrogen activation of gene expression is the interaction of the estrogen receptor with a specific nucleotide sequence [estrogen responsive element (ERE)]. Previously, we found that the estrogen receptor binds preferentially and with high affinity to the lower strand of the rat prolactin imperfect ERE which contains tertiary structure (Lannigan DA and Notides AC, Proc Natl Acad Sci USA 86: 863-867, 1989). Using perfect and imperfect EREs from the upstream region of the chicken vitellogenin II gene, we have now extended our findings and have determined that the estrogen receptor preferentially interacts with either perfect or imperfect EREs which contain tertiary structure. A similar structure is present in a synthetic 42 bp oligonucleotide corresponding to the lower strand of a perfect ERE with flanking sequences from the rat prolactin ERE. Moreover, deviations from the ERE consensus sequence decrease the binding of the estrogen receptor to the tertiary-structured ERE. We also have determined that ERE flanking sequences contribute to the affinity of the receptor for the tertiary-structured ERE. Furthermore, ERE flanking sequences can influence the types of interactions that the estrogen receptor makes with the tertiary-structured ERE.     

Functional specificity of the mediator systems as the basis for neostriatum polyfunctional properties

Following intrastriatal administration of dopamine-, GABA-, and encephalinergic drugs, the changes occurring in the rat behaviour were associated with a concrete transmitter system's activity and neostriatum polyfunctionality (and not an unspecific nature).     

Coping as a mediator of emotion.

There is widespread conviction among health care professionals that coping affects emotion. Yet theory and research have traditionally emphasized the effects of emotion on coping. The present research addresses this imbalance by evaluating the extent to which coping mediated emotions during stressful encounters in two Caucasian, community-residing samples. Subjects' recently experienced stressful encounters, the ways they coped with the demands of those encounters, and the emotions they experienced during two stages of those encounters were assessed repeatedly. The extent to which eight forms of coping mediated each of four sets of emotions was evaluated with a series of hierarchical regression analyses (of residuals). Coping was associated with changes in all four sets of emotions, with some forms of coping associated with increases in positive emotions and other forms associated with increases in negative emotions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modulation of intestinal estrogen receptor by ovariectomy, estrogen and growth hormone

Ovarian hormone deficiency decreases and estrogen (E2) and growth hormone (GH) administrations increase intestinal absorption of calcium (Ca++). However, the underlying mechanisms are uncertain. To examine whether alterations in the binding characteristics of intestinal estrogen receptors (ERs) are involved, we developed and validated methods for simultaneous measurement of intestinal ERs in cytosolic and nuclear fractions and applied these techniques to four groups of female rats: sham-operated, ovariectomized (Ovx), Ovx + 5 micrograms E2/kg b.wt./day and Ovx + 8 mg GH/kg. b.wt./day. All animals were killed on day 21, and mucosal cells harvested from the duodenum for ER determination. The cytosolic and nuclear ERs were 117.2 +/- 2.7 fmol/mg protein and 64.9 +/- 1.2 fmol/mg DNA, respectively, in sham-operated rats and decreased by 16.1% and 17.0% to 98.4 +/- 1.7 fmol/mg protein and 53.8 +/- 1.3 fmol/mg DNA, respectively in Ovx rats (P < .001). E2 therapy prevented completely the decrease in cytosolic and nuclear ERs that occurred in Ovx rat (126.1 +/- 2.9 fmol/mg protein and 68.0 +/- 3.0 fmol/mg DNA, respectively, in the E2-treated group). Similarly, GH administration prevented the decrease in cytosolic and nuclear ERs that resulted from ovariectomy (119.2 +/- 3.2 fmol/mg protein and 63.4 +/- 1.3 fmol/mg DNA, respectively, in the GH-treated group). The Kd of nuclear ER-ligand complex was 2.0 +/- 0.03 nM in sham-operated rats and was slightly modulated by Ovx, E2 and GH (3.3 +/- 0.02, 2.33 +/- 0.09 and 2.23 +/- 0.04 nM, respectively, P < .001), but the Kd of cytosolic ER-ligand complex was not altered by Ovx, E2 or GH. Our findings indicate that E2 deficiency down-regulates, whereas E2 and GH administrations up-regulate intestinal ERs and prevent ovariectomy-induced decrease in receptor binding affinity. We conclude that E2 deficiency, E2 and GH may modulate intestinal Ca++ absorption, in part, by altering the abundance and binding characteristics of intestinal ERs.     

Epidermal growth factor receptor as a mediator of developmental toxicity of dioxin in mouse embryonic teeth

We have previously shown that dioxins at prevailing levels in mothers' milk may cause mineralization defects in the developing teeth of their children. Developmental dental defects have also been reported in rhesus macaques and rats experimentally exposed to dioxin. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a potent modulator of epithelial cell growth and differentiation. To clarify whether epidermal growth factor receptor (EGFR), implicated in the mediation of the developmental toxicity of TCDD, is involved in dental toxicity, we cultured embryonic molar teeth from EGFR-deficient mice with TCDD, epidermal growth factor (EGF), and both agents in combination. In teeth of the normal embryos, TCDD caused depolarization of odontoblasts and ameloblasts. Consequently, the dentin matrix failed to undergo mineralization, the enamel matrix was not deposited, and cuspal morphology was disrupted. In teeth of the null mutant embryos, only the cuspal contour was mildly modified. EGF alone retarded the molar tooth development of normal embryos, but not that of EGFR-deficient embryos. When coadministered with TCDD, EGF for the most part prevented the adverse effects of TCDD on teeth of the normal embryos. These results show that the interference of TCDD with mouse molar tooth development in vitro involves EGFR signaling. Thus, EGFR may also play a role in the developmental defects that dioxins cause in human teeth. Because EGFR is widely expressed in developing organs, EGFR signaling may even be of general relevance in the mediation of the developmental toxicity of TCDD.     

Selective estrogen receptor modulators as a new concept in preventing health risks of menopause

Long-term estrogen deficiency after menopause is responsible for different disorders, which not only make the quality of life in the older age worse but also are the major causes of women's mortality. It is especially the case for cardiovascular disease, osteoporosis and dementia. The risk for these disorders can significantly be reduced by hormone replacement therapy (HRT). Unfortunately, the mean duration of the postmenopausal administration of HRT is too short to demonstrate its efficacy in preventing the mentioned diseases. In this review the new therapeutic possibilities are discussed, called selective estrogen receptor modulators (SERMs). These structurally heterogeneous compounds interact with estrogen receptors and act as either estrogen-agonists or--antagonists according to the type of organ and physiological context (i.e., dose, target tissue and hormone concentration in the tissue). The evaluation of the effects of these compounds led to the better understanding of both antiestrogens and the whole steroid signaling system. The research of the clinical properties of SERM showed their potential benefit in the long-term care of the women in their non-reproductive period of life and demonstrated the possibility to overcome some drawbacks of HRT.     

Tau self-association: stabilization with a chemical cross-linker and modulation by phosphorylation and oxidation state

tau is a major component of paired helical filaments found in the neurofibrillary tangles of Alzheimer's diseased brain. However, the mechanism or mechanisms responsible for the association of tau to form these aggregates remains unknown. In this study, the role of intermolecular disulfide bonds in the formation of higher order oligomers of bovine tau and the human recombinant tau isoform T3 was examined using the chemical cross-linking agent disuccinimidylsuberate (DSS). In addition, the role of phosphorylation and oxidation state on the in vitro self-association of tau was studied using this experimental model. Stabilization of tau-tau interactions with DSS indicated that intermolecular disulfide bonds probably play a predominant role in dimer formation, but the formation of higher order oligomers of tau cannot be attributed to these bonds alone. tau-tau interactions were significantly decreased either by blocking Cys residues or by exposing the tau to a reducing (nitrogen and dithiothreitol), instead of an oxidizing, environment. tau self-association was also significantly decreased by prior phosphorylation with calcium/calmodulin-dependent protein kinase II. Phosphorylation by cyclic AMP-dependent protein kinase or dephosphorylation by alkaline phosphatase did not alter tau self-assembly. These data suggest a role for several factors that may modulate tau self-association in vivo.     

Triazolam-induced modulation of muscarinic acetylcholine receptor in living brain slices as revealed by a new positron-based imaging technique

The effect of triazolam, a potent benzodiazepine (BZ) agonist, on muscarinic acetylcholinergic receptor (mAChR) binding was investigated in living brain slices by use of a novel positron-based imaging technique. Fresh rat brain slices were incubated with [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a mAChR antagonist, in oxygenated Krebs-Ringer solution at 37 degrees C. During incubation, time-resolved imaging of [11C]NMPB binding in the slices was constructed on the storage phosphor screens. Addition of triazolam (1 microM) plus muscimol (30 microM), a GABA(A) receptor agonist, to the incubation mixture decreased the specific binding of [11C]NMPB. Ro15-1788, a BZ receptor antagonist, prevented this effect, indicating that the effect was exerted through the GABA(A)/BZ receptor complex. These results demonstrated that stimulation of the GABA(A)/BZ receptor lowers the affinity of the mAChR for its ligand, which may underlie the BZ-induced amnesia, a serious clinical side effect of BZ. No such effect in the P2-fraction instead implies that the integrity of the neuronal cells and/or their environment is prerequisite for the modulation of mAChR by GABA(A)/BZ stimulation.     

Partner support as a mediator of intervention outcome

Following up on previous work demonstrating that an intervention with at-risk mothers made a positive impact on the quality of mothers' partner support, responsiveness to the needs of the child, the child's expectation of being cared for, and child's response to a brief separation, the present paper examines whether quality of the mother's partner support mediated the impact of the intervention on these outcomes.     

The interaction of human estrogen receptor with DNA is modulated by receptor-associated proteins

To better define the role of accessory protein as mediators of estrogen receptor (ER) function, we have used immuno-, steroid-, and site-specific DNA-affinity chromatography to identify and characterize proteins that associate with ER in extracts from ER-expressing Chinese hamster ovary (CHO-ER) cells. Two associated proteins [70 and 55 kilodaltons (kDa)] were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis silver stain analysis of all three column eluates. Two additional proteins (45 and 48 kDa) were preferentially retained by the ER-specific DNA affinity column. The 70-kDa protein was subsequently identified by Western blot analysis as a heat shock protein (hsp70). The 55-kDa protein was identified by N-terminal microsequencing as a member of the protein disulfide isomerase family. The 48- and 45-kDa proteins remain unidentified. To determine the possible differential effects of estrogen agonists and antagonists on human (h) ER interaction with these proteins, CHO-ER cells were labeled with [35S]methionine in the presence of estradiol, 4-hydroxytamoxifen (OH-Tam; partial agonist/antagonist), or ICI 182,780 (complete antagonist). None of these ligands altered the pattern of associated proteins when hER complexes were isolated by adsorption to the vitellogenin A2 estrogen response element (ERE). However, when hER was isolated by immunoadsorption, a reduction in the level of associated hsp70 was observed following treatment with estradiol or OH-Tam, compared to no treatment or treatment with ICI 182,780. By gel retardation analysis, maximal interaction of affinity-purified hER with ERE occurred in the presence of all four associated proteins. Removal of the 48- and 45-kDa proteins and/or hsp70 resulted in a decrease in hER/ERE association, which could be restored by the addition of purified hsp70 and/or a mixture of the 48- and 45-kDa proteins. The increased stability of the restored complex was due primarily to an increase in the association rate of hER with ERE. These results suggest that accessory proteins may be required for maximal ER interaction with EREs and that estrogens and estrogen antagonists may promote differential retention of hsp70 in the presence or absence of a specific ERE.     

Up-regulation of estrogen receptor mRNA and estrogen receptor activity by estradiol in liver of rainbow trout and other teleostean fish

[125I]- and [123I]NNC 13-8241 were prepared from the trimethyltin precursor and radioactive iodide using the chloramine-T method. The total radiochemical yields of [125I]- and [123I]NNC 13-8241 were 60-70% and 40-50% respectively, with radiochemical purity higher than 98%. In binding studies with [125I]NNC 13-8241 in rats in vitro and in vivo a high uptake of radioactivity was demonstrated in brain regions known to have a high density of benzodiazepine (BZ) receptors such as the occipital and frontal cortex. SPECT examination with [123I]NNC 13-8241 in a Cynomolgus monkey demonstrated a high uptake of radioactivity in the occipital and frontal cortex. After displacement with flumazenil radioactivity in these brain regions was reduced to the level of a central region including the pons. Four hours after injection about 80% of the radioactivity in monkey plasma represented unchanged radioligand. This low degree of metabolism indicates that NNC 13-8241 is metabolically more stable than the radioligands hitherto developed for imaging of BZ-receptors in the primate brain.     

Mood as a mediator of place dependent memory.

Converging evidence from 3 studies suggests that how well information transfers from one environment to another depends on how similar the environments feel rather than on how similar they look. Thus, even when target events are encoded and retrieved in the same physical setting, memory performance suffers if the attending affective states differ. Conversely, a change in environment produces no performance decrement if, whether by chance (Experiments 1 and 2) or by design (Experiment 3), the mood at encoding matches the mood at retrieval. These observations imply that place dependent effects are mediated by alterations in affect or mood, and that data that appear on the surface to demonstrate place dependent memory may, at a deeper level, denote the presence of mood dependent memory. Discussion focuses on prospects for future research aimed at clarifying the relations among moods, places, and memory. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Decrease of hormone binding capacity of estrogen receptor by calcium

We have addressed the question as to whether calcium may modify the [3H]estradiol ([3H]E2) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues. Ca++ (0.1-10 mM) always produced an important loss of [3H]E2 binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [125I]Z-17 alpha-(2-iodovinyl)-11 beta-chloromethyl estradiol-17 beta (an compound with very high selectivity for ER) as well as [125I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca(++)-induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [3H]E2 binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca++ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca++ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.     

Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of both the redox active selenocysteine and its neighboring cysteine residue

The immunostimulatory dinitrohalobenzene compound 1-chloro-2, 4-dinitrobenzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase (TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in the modified enzyme (Arnér, E. S. J., Bj?rnstedt, M., and Holmgren, A. (1995) J. Biol. Chem. 270, 3479-3482). Here we have further analyzed the reactivity with the enzyme of DNCB and analogues with varying immunomodulatory properties. We have also identified the reactive residues in bovine thioredoxin reductase, recently discovered to be a selenoprotein. We found that 4-vinylpyridine competed with DNCB for inactivation of TrxR, with DNCB being about 10 times more efficient, and only alkylation with DNCB but not with 4-vinylpyridine induced an NADPH oxidase activity. A number of nonsensitizing DNCB analogues neither inactivated the enzyme nor induced any NADPH oxidase activity. The NADPH oxidase activity of TrxR induced by dinitrohalobenzenes generated superoxide, as detected by reaction with epinephrine (the adrenochrome method). Addition of superoxide dismutase quenched this reaction and also stimulated the NADPH oxidase activity. By peptide analysis using mass spectrometry and Edman degradation, both the cysteine and the selenocysteine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where Sec indicates selenocysteine) were determined to be dinitrophenyl-alkylated upon incubation of native TrxR with NADPH and DNCB. A model for the interaction between TrxR and dinitrohalobenzenes is proposed, involving a functional FAD in the alkylated TrxR generating an anion nitroradical in a dinitrophenyl group, which in turn reacts with oxygen to generate superoxide. Production of reactive oxygen species and inhibited reduction of thioredoxin by the modified thioredoxin reductase after reaction with dinitrohalobenzenes may play a major role in the inflammatory reactions provoked by these compounds.     

G-Protein-coupled modulation of presynaptic calcium currents and transmitter release by a GABAB receptor

Presynaptic GABAB receptors play a regulatory role in central synaptic transmission. To elucidate their underlying mechanism of action, we have made whole-cell recordings of calcium and potassium currents from a giant presynaptic terminal, the calyx of Held, and EPSCs from its postsynaptic target in the medial nucleus of the trapezoid body of rat brainstem slices. The GABAB receptor agonist baclofen suppressed EPSCs and presynaptic calcium currents but had no effect on voltage-dependent potassium currents. The calcium current-EPSC relationship measured during baclofen application was similar to that observed on reducing [Ca2+]o, suggesting that the presynaptic inhibition generated by baclofen is caused largely by the suppression of presynaptic calcium influx. Presynaptic loading of the GDP analog guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) abolished the effect of baclofen on both presynaptic calcium currents and EPSCs. The nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) suppressed presynaptic calcium currents and occluded the effect of baclofen on presynaptic calcium currents and EPSCs. Photoactivation of GTPgammaS induced an inward rectifying potassium current at the calyx of Held, whereas baclofen had no such effect. We conclude that presynaptic GABAB receptors suppress transmitter release through G-protein-coupled inhibition of calcium currents.