Search PubMed⌕ Search

Biomedical subjects

C L Smith

Publications and source records attributed to C L Smith.

At least 37 records · Page 2Linked to original sources
PubMed2001

Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor alpha-coactivator complexes in living cells.

Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor alpha (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-tagged lac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integrated lac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP-SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP-SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP-SRC-1, while antagonist additions diminish YFP-SRC-1-CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER-SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP-SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

Amino Acid Motifs↗
PubMed2001

A-ring reduced metabolites of 19-nor synthetic progestins as subtype selective agonists for ER alpha.

It has previously been demonstrated that 19-nor contraceptive progestins undergo in vivo and in vitro enzyme-mediated A-ring double bond hydrogenation. Bioconversion of 19-nor progestins to their corresponding tetrahydro derivatives results in the loss of progestational activity and acquisition of estrogenic activities and binding to the ER. Herein, we report subtype-selective differences in ligand binding and transcriptional potency of nonphenolic synthetic 19-nor derivatives between ER alpha and ER beta. In this study, we have examined both ER- and PR-mediated transcriptional activity of a number of A-ring chemically reduced derivatives of norethisterone and Gestodene. Double bond hydrogenation decreased the transcriptional potency of norethisterone and Gestodene through both PR isoforms with a 100- to 1,000-fold difference, respectively. In terms of the effects of norethisterone and Gestodene and their corresponding 5 alpha-dihydro (5 alpha-norethisterone and 5 alpha-Gestodene), or 3 alpha,5 alpha-tetrahydro or 3 beta,5 alpha-tetrahydro derivatives (3 alpha,5 alpha-norethisterone/3 alpha,5 alpha-Gestodene and 3 beta,5 alpha-norethisterone/3beta,5 alpha-Gestodene, respectively) on estrogen-mediated transcriptional regulation, the 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene showed the highest induction when HeLa cells were transiently transfected with an expression vector for ER alpha. This activity could be inhibited with tamoxifen. These compounds did not activate gene transcription via ER beta, and none of them showed antagonistic activities through either ER subtype. The 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene were active in other cells in addition to HeLa cells and activated reporter expression through the oxytocin promoter. In summary, two ER alpha selective agonists have been identified. These compounds, with ER alpha vs. ER beta selective agonist activity, may be useful in evaluating the distinct role of these receptors as well as in providing useful insights into ER action.

Animals↗
PubMed2001

New antiprogestins with partial agonist activity: potential selective progesterone receptor modulators (SPRMs) and probes for receptor- and coregulator-induced changes in progesterone receptor induction properties.

A pharmacologically relevant property of steroid hormone-regulated gene induction is the partial agonist activity of antisteroid complexes. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of progesterone receptor (PR), for different progesterone-responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine whether the partial agonist activity of PR complexes can be modified by changing concentrations of receptor or coregulator, as we have recently demonstrated for glucocorticoid receptors (GRs). Because GR and coregulator concentrations simultaneously altered the position of the physiologically relevant dose-response curve, and associated EC(50), of GR-agonist complexes, we also examined this phenomenon with PR. We find that elevated PR or transcriptional intermediary factor 2 (TIF2) concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and the EC(50) of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT (silencing mediator for retinoid and thyroid receptors) and NCoR (nuclear receptor corepressor) each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC(50) and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the magnitude of coregulator effects on PR induction properties are often not identical for agonists and the new antagonists, suggesting subtle mechanistic differences. These properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties.

Animals↗
PubMed2001

Making it count.

Explore the source record for details and available documents.

Health Policy↗
PubMed2001

An evaluation of the physicochemical risk for renal stone disease during pregnancy.

Seventeen subjects were studied during the third trimester of pregnancy (PG) and post partum (NPG) to evaluate the effect of pregnancy on the physicochemical risk of renal stone disease. Levels of urinary saturation for calcium oxalate (CaOx), brushite (Br), uric acid (UA), and monosodium urate (NaU) were determined as well as urinary excretions of stone-forming elements. In addition to urinary calcium excretion, assessment of calcium metabolism included serum calcium and parathyroid hormone. Urinary calcium excretion was 251 +/- 127 mg/day during pregnancy and 121 +/- 67 mg/day post partum (p < 0.001). This was associated with a higher intake of dietary calcium and altered renal handling of calcium with an increase in the filtered load and a decrease in renal tubular reabsorption. The increase in urinary calcium resulted in a higher level of saturation of the urine for calcium oxalate (NPG 2.1 +/- 1.0 vs PG 3.0 +/- 1.1, p < 0.02) and brushite (NPG 1.2 +/- 0.9 vs PG 1.9 +/- 1.1, p < 0.05) compatible with an increased risk of stone formation.

Adult↗
PubMed2000

A shift in the phenotype of melan-A-specific CTL identifies melanoma patients with an active tumor-specific immune response.

In a significant proportion of melanoma patients, CTL specific for the melan-A(26/7-35) epitope can be detected in peripheral blood using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic markers to characterize melan-A tetramer(+) cells in both normal individuals and melanoma patients, and correlated these markers with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramer(+) cells in peripheral blood fell into two groups. Seven of thirteen patients had a CCR7(+) CD45R0(-) CD45RA(+) phenotype, the same as that found in some healthy controls, and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A tetramer(+) cells were shifted toward a CCR7(-) CD45R0(+) CD45RA(-) phenotype, and responses to melan-A peptide could be readily demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays.

Adult↗
PubMed2000

Effects of antiandrogens on chromatin remodeling and transcription of the integrated mouse mammary tumor virus promoter.

Inhibition of the ligand-activated androgen receptor (AR) by antiandrogens plays an important role in the treatment of various hyperandrogenic disorders including prostate cancer. However, the molecular mechanisms of antiandrogen activity in vivo remain unclear. In this study we analyzed the effects of cyproterone acetate (CPA), flutamide (F), and hydroxyflutamide (OHF) on transcriptional activation and chromatin remodeling of the genomically integrated mouse mammary tumor virus (MMTV) promoter. This promoter has provided an excellent model system to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The MMTV hormone response element is positioned on a phased nucleosome, which becomes remodeled in response to steroids. We utilized this model system in mouse L-cell fibroblasts that contain a stably integrated MMTV promoter. In these cells, dihydrotestosterone (DHT) induced a large increase of AR protein levels that correlated with transcriptional activation and chromatin remodeling of the MMTV promoter. Coadministration of DHT and CPA or DHT and OHF in these cells inhibited the increase of AR levels, which resulted in a strong blockage of transcriptional activation and chromatin remodeling of the MMTV promoter. In contrast, F had no significant influence on these activities. We conclude that a major portion of the antiandrogenic effects of CPA and OHF in vivo are mediated by the reduction of AR levels.

Androgen Antagonists↗
PubMed2000

Oligonucleotide immobilization on micropatterned streptavidin surfaces.

We describe a simple procedure for photolithographic patterning of streptavidin on silicon substrates. Long wavelength UV (365 nm) light was used to direct the covalent attachment of photoactivatable biotin onto silylated silicon wafers. Fluorescently labeled streptavidin was found to bind only in areas exposed to the light. We used this procedure to selectively pattern streptavidin inside microwells etched in silicon, and we investigated the binding characteristics of biotinylated oligonucleotides of lengths, n = 16, 54 and 99 bases. The binding curves were found to fit the functional form of the Langmuir isotherm, with binding saturation proportional to n(-3/4).

Biotin↗
PubMed2000

Evaluation of newer treatment interventions for psychotic depression.

Psychotic depression is characterized by greater severity, higher rate of recurrence, greater incapacitation, more frequent hospitalization, and longer episodes than nonpsychotic depression. The use of combined tricyclic antidepressants (TCAs) and neuroleptic therapy, as well as electroconvulsive therapy, has proven effectiveness in the treatment of psychotic depression. Although it is limited, evidence for efficacy of selective serontonin reuptake inhibitors both alone and in combination with antipsychotics in psychotic depression suggests that these strategies may provide a desirable alternative to the traditional TCA- neuroleptic therapy. These treatments, in addition to the continual introduction of new psychotropic agents suggest that the prospect of future research in this area is promising.

Antidepressive Agents↗
PubMed2000

Indentation of an osteochondral repair: sensitivity to experimental variables and boundary conditions.

The sensitivity of the affects of indenter radius, defect depth, cartilage permeability and flow boundary conditions, on the indentation testing of a repairing osteochondral defect was investigated. Since the boundary condition on the flow across the cartilage repair-subchondral bone interface is not known, the effects of two different conditions were investigated: free-flow and no-flow. A poroelastic finite element model of an osteochondral defect at different stages of the repair process was developed using dimensions typical of the rabbit knee. Results showed when the radius of the indenter was much less than the thickness of the cartilage the sensitivity of the indentation displacement to flow boundary conditions decreased. Simulated indentation displacement was insensitive to bone regeneration up to 50% of the initial defect depth, which suggests that only the properties of the material in the upper-half of the defect are being evaluated. Small variations in permeability had little affect on the simulated indentation. In a fully repaired defect, the simulated indentation is independent of the boundary condition. However, while the defect is in the process of repair and the regenerated cartilage is deeper than the host, indentation is sensitive to the flow boundary condition. Based on these results, and feasible experimental conditions, we conclude that the boundary condition on the repair-subchondral bone interface must be known in all cases except when the defect approaches full repair, if accurate estimates of material properties are to be obtained from indentation.

Animals↗
PubMed2000

Dog model of therapeutic ketosis induced by oral administration of R,S-1,3-butanediol diacetoacetate.

A high-fat, almost carbohydrate-free diet is used in children with intractable epilepsy to help control seizures by inducing a permanent state of ketosis. Esters of ketone bodies have been previously studied for their potential as parenteral and enteral nutrients. We tested in conscious dogs whether ketosis could be induced by repeated ingestion of R,S-1,3-butanediol diacetoacetate with or without carbohydrates. This ester is a water-soluble precursor of ketone bodies. Two constraints were imposed on this preclinical study: The rate of ester administration was limited to one half of the daily caloric requirement and to one half of the capacity of the liver to oxidize butanediol derived from ester hydrolysis. Under these conditions, the level of ketosis achieved in this dog model (0.8 mM) was lower than the level measured in children whose seizures were controlled by the ketogenic diet (1-3 mM). However, because humans may have a lower capacity for ketone body utilization than dogs, the doses of R,S-butanediol diacetoacetate used in the present study might induce higher average ketone body concentrations in humans than in dogs.

Journal Article↗
PubMed2000

Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control.

Cdc6/Cdc18 is a conserved and essential component of prereplication complexes. The 2.0 A crystal structure of an archaeal Cdc6 ortholog, in conjunction with a mutational analysis of the homologous Cdc18 protein from Schizosaccharomyces pombe, reveals novel aspects of Cdc6/Cdc18 function. Two domains of Cdc6 form an AAA+-type nucleotide binding fold that is observed bound to Mg.ADP. A third domain adopts a winged-helix fold similar to known DNA binding modules. Sequence comparisons show that the winged-helix domain is conserved in Orc1, and mutagenesis data demonstrate that this region of Cdc6/Cdc18 is required for function in vivo. Additional mutational analyses suggest that nucleotide binding and/or hydrolysis by Cdc6/Cdc18 is required not only for progression through S phase, but also for maintenance of checkpoint control during S phase.

Alleles↗
PubMed2000

The 26S proteasome is required for estrogen receptor-alpha and coactivator turnover and for efficient estrogen receptor-alpha transactivation.

Estrogen receptor-alpha (ER alpha) is downregulated in the presence of its cognate ligand, estradiol (E2), through the ubiquitin proteasome pathway. Here, we show that ubiquitin proteasome function is required for ER alpha to serve as a transcriptional activator. Deletion of the last 61 amino acids of ER alpha, including residues that form helix 12, abolishes ligand-mediated downregulation of the receptor as do point mutations in the ligand binding domain that impair coactivator binding. In addition, coactivators also are subject to degradation by the 26S proteasome, but their intrinsic transcriptional activity is not affected. These data provide evidence that protein interactions with ER alpha coactivator binding surfaces are important for ligand-mediated receptor down-regulation and suggest that receptor and coactivator turnover contributes to ER alpha transcriptional activity.

Amino Acid Sequence↗
PubMed2000

Reliability and validity of DSM-IV axis V.

OBJECTIVE: The authors investigated the reliability and convergent and discriminant validity of the DSM-IV Global Assessment of Functioning Scale and two experimental DSM-IV axis V global rating scales, the Global Assessment of Relational Functioning Scale and the Social and Occupational Functioning Assessment Scale. METHOD: Forty-four patients admitted to a university-based outpatient community clinic were rated by trained clinicians on the three DSM-IV axis V scales. Patients also completed self-report measures of DSM-IV symptoms as well as measures of relational, social, and occupational functioning. RESULTS: The Global Assessment of Functioning Scale, Global Assessment of Relational Functioning Scale, and Social and Occupational Functioning Assessment Scale all exhibited very high levels of interrater reliability. Factor analysis revealed that the Global Assessment of Relational Functioning Scale and the Social and Occupational Functioning Assessment Scale are each more related to the Global Assessment of Functioning Scale individually than they are to each other. The Global Assessment of Functioning Scale was significantly related to concurrent patient responses on the SCL-90-R global severity index. The Social and Occupational Functioning Assessment Scale was significantly related to concurrent patient responses on the SCL-90-R global severity index and to a greater degree with both the Social Adjustment Scale global score and the Inventory of Interpersonal Problems total score. Although the Global Assessment of Relational Functioning Scale was not significantly related to any of the three self-report measures, it was related to the presence of clinician-rated axis II pathology. CONCLUSIONS: The three axis V scales can be scored reliably. The Global Assessment of Relational Functioning Scale and the Social and Occupational Functioning Assessment Scale evaluate different constructs. These findings support the validity of the Global Assessment of Functioning Scale as a scale of global psychopathology; the Social and Occupational Functioning Assessment Scale as a measure of problems in social, occupational, and interpersonal functioning; and the Global Assessment of Relational Functioning Scale as an index of personality pathology. The authors discuss further refinement and use of the three axis V measures in treatment research.

Adult↗
PubMed2000

Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1.

We have analyzed ligand-dependent, subnuclear movements of the estrogen receptor-alpha (ERalpha) in terms of both spatial distribution and solubility partitioning. Using a transcriptionally active green fluorescent protein-ERalpha chimera (GFP-ERalpha), we find that 17beta-estradiol (E2) changes the normally diffuse nucleoplasmic pattern of GFP-ERalpha to a hyperspeckled distribution within 10-20 min. A similar reorganization occurs with the partial antagonist 4-hydroxytamoxifen; only a subtle effect was observed with the pure antagonist ICI 182,780. To examine the influence of ligand upon ERalpha association with nuclear structure, MCF-7 cells were extracted to reveal the nuclear matrix (NM). Addition of E2, 4-hydroxytamoxifen, or ICI 182,780 causes ERalpha to partition with the NM-bound fraction on a similar time course (10-20 min) as the spatial reorganization suggesting that the two events are related. To determine the effects of E2 on the redistribution and solubility of GFP-ERalpha, individual cells were directly examined during both hormone addition and NM extraction and showed that GFP-ERalpha movement and NM association were coincident. Colocalization experiments were performed with antibodies to identify sites of transcription (RNA pol Ilo) and splicing domains (SRm160). Using E2 treated MCF-7 cells, minor overlap was observed with transcription sites and a small amount of the total ERalpha pool. Experiments performed with bioluminescent derivatives of ERalpha and steroid receptor coactivator-1 (SRC-1) demonstrated both proteins colocalize to the same NM-bound foci in response to E2 but not the antagonists tested. Deletion mutagenesis and in situ analyses indicate intranuclear colocalization requires a central SRC-1 domain containing LXXLL motifs. Collectively, our data suggest that ERalpha transcription function is dependent upon dynamic early events including intranuclear rearrangement, NM association, and SRC-1 interactions.

Antibodies, Monoclonal↗
PubMed2000

Characterization of transiently and constitutively expressed progesterone receptors: evidence for two functional states.

Activated steroid receptors induce chromatin remodeling events in the promoters of some target genes. We previously reported that transiently expressed progesterone receptor (PR) cannot activate mouse mammary tumor virus (MMTV) promoter when it adopts the form of ordered chromatin. However, when expressed continuously, the PR acquires this ability. In this study we explored whether this gain of function occurs through alterations in nucleoprotein structure at the MMTV promoter or through changes in receptor status. We observed no major structural differences at the MMTV promoter in the presence of constitutively expressed PR and found its mechanism of activation to be very similar to that of the glucocorticoid receptor (GR). However, a systematic comparison of the functional behavior of the transiently and constitutively expressed PR elucidated significant differences. The transiently expressed PR is activated in the absence of ligand by cAMP and by components in FBS and has significantly increased sensitivity to progestins. In contrast, the constitutively expressed PR is refractory to activation by cAMP and serum and has normal sensitivity to its ligand. In addition, while the PR is localized to the nucleus in both cases, a significant fraction of the transiently expressed PR is tightly bound to the nucleus even in the absence of ligand, while the majority of constitutively expressed PR is not. These results strongly suggest that the PR undergoes processing in the cell subsequent to its initial expression and that this processing is important for various aspects of its function, including its ability to productively interact with target genes that require chromatin remodeling for activation.

Animals↗
PubMed2000

Molecular mechanisms of selective estrogen receptor modulator (SERM) action.

In females, estrogens play a key role in reproduction and have beneficial effects on the skeletal, cardiovascular, and central nervous systems. Most estrogenic responses are mediated by estrogen receptors (ERs), either ER alpha or ER beta, which are members of the nuclear receptor superfamily of ligand-dependent transcription factors. Selective estrogen receptor modulators (SERMs) are ER ligands that in some tissues act like estrogens, but block estrogen action in others. Thus, SERMs may exhibit an agonistic or antagonistic biocharacter depending on the context in which their activity is examined. For example, the SERMs tamoxifen and raloxifene both exhibit ER antagonist activity in breast and agonist activity in bone, but only tamoxifen manifests agonist activity in the uterus. Numerous studies have examined the molecular basis for SERM selectivity. Collectively they indicate that different ER ligands induce distinct structural changes in the receptor that influence its ability to interact with other proteins (e.g., coactivators or corepressors) critical for the regulation of target gene transcription. The relative expression of coactivators and corepressors, and the nature of the ER and of its target gene promoter affect SERM biocharacter. Taken together, SERM selectivity reflects the diversity of ER forms and coregulators, cell type differences in their expression, and the diversity of ER target genes. This model provides a basis for understanding the molecular mechanisms of SERM action, and should help identify new SERMs with enhanced tissue or target gene selectivity.

Humans↗