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P Webb

Publications and source records attributed to P Webb.

At least 37 records · Page 2Linked to original sources
PubMed2000

The nuclear receptor corepressor (N-CoR) contains three isoleucine motifs (I/LXXII) that serve as receptor interaction domains (IDs).

Unliganded thyroid hormone receptors (TRs) repress transcription through recruitment of corepressors, including nuclear receptor corepressor (N-CoR). We find that N-CoR contains three interaction domains (IDs) that bind to TR, rather than the previously reported two. The hitherto unrecognized ID (ID3) serves as a fully functional TR binding site, both in vivo and in vitro, and may be the most important for TR binding. Each ID motif contains a conserved hydrophobic core (I/LXXII) that resembles the hydrophobic core of nuclear receptor boxes (LXXLL), which mediates p160 coactivator binding to liganded nuclear receptors. Although the integrity of the I/LXXII motif is required for ID function, substitution of ID isoleucines with leucines did not allow ID peptides to bind to liganded TR, and substitution of NR box leucines with isoleucines did not allow NR box peptides to bind unliganded TR. This indicates that the binding preferences of N-CoR for unliganded TR and p160s for liganded TR are not dictated solely by the identity of conserved hydrophobic residues within their TR binding motifs. Examination of sequence conservation between IDs, and mutational analysis of individual IDs, suggests that they are comprised of the central hydrophobic core and distinct adjacent sequences that may make unique contacts with the TR surface. Accordingly, a hybrid peptide that contains distinct adjacent sequences from ID3 and ID1 shows enhanced binding to TR.

Amino Acid Motifs↗
PubMed1999

Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta.

Neutrophils are short-lived leukocytes that die by apoptosis. Whereas stress-induced apoptosis is mediated by the p38 mitogen-activated protein (MAP) kinase pathway (Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389-8397), signals regulating spontaneous neutrophil apoptosis have not been fully determined. In this study we found increased activation of protein kinase C (PKC)-beta and -delta in neutrophils undergoing spontaneous apoptosis, but we show that only activation of PKC-delta was directly involved in the induction of apoptosis. PKC-delta can be proteolytically activated by caspase 3. We detected the 40-kDa caspase-generated fragment of PKC-delta in apoptotic neutrophils and showed that the caspase 3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone prevented generation of the 40-kDa PKC-delta fragment and delayed neutrophil apoptosis. In a cell-free system, removal of PKC-delta by immunoprecipitation reduced DNA fragmentation, whereas loss of PKC-alpha, -beta, or -zeta had no significant effect. Rottlerin and LY379196 inhibit PKC-delta and PKC-beta, respectively. Only Rottlerin was able to delay neutrophil apoptosis. Inhibitors of MAP-ERK kinase 1 (PD98059) or p38 MAP kinase (SB202190) had no effect on neutrophil apoptosis, and activation of p42/44 and p38 MAP kinase did not increase in apoptotic neutrophils. We conclude that spontaneous neutrophil apoptosis involves activation of PKC-delta but is MAP kinase-independent.

Apoptosis↗
PubMed1999

Estradiol repression of tumor necrosis factor-alpha transcription requires estrogen receptor activation function-2 and is enhanced by coactivators.

The tumor necrosis factor-alpha (TNF-alpha) promoter was used to explore the molecular mechanisms of estradiol (E(2))-dependent repression of gene transcription. E(2) inhibited basal activity and abolished TNF-alpha activation of the TNF-alpha promoter. The E(2)-inhibitory element was mapped to the -125 to -82 region of the TNF-alpha promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) beta is more potent than ERalpha at repressing the -1044 TNF-alpha promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERbeta, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E(2)-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Salivary gland neoplasms with basaloid cell features: report of two cases diagnosed by fine-needle aspiration cytology.

Basal-cell adenoma and basal-cell adenocarcinoma of the salivary gland are rare tumors. Fine-needle aspiration cytology of these tumors, particularly those of basal-cell adenocarcinoma, has rarely been described in the literature. In this report, we describe the clinical, cytomorphologic, histopathologic, and immunohistochemical features of basal-cell adenoma and its malignant counterpart, basal-cell adenocarcinoma, in 2 patients. Fine-needle aspiration specimens from both tumors contained abundant cohesive groups of neoplastic cells. Basaloid cells were prominent in both tumors; however, there were significant cytologic atypia, hyperchromasia, and increased nuclear-to-cytoplasmic ratio in basal-cell adenocarcinoma. Review of the literature and cytomorphologic distinction between both tumors and others are discussed.

Adenocarcinoma↗
PubMed1999

Splenic peliosis: a rare complication following liver transplantation.

In this article, we report a rare case of isolated splenic peliosis in an individual who had recently undergone liver transplantation. The disorder had remained clinically and radiologically undiagnosed until he suffered a traumatic rupture of the affected organ. The relevant literature on this topic is briefly reviewed.

Biopsy↗
PubMed1999

Mutation inhibition by beta-estradiol after low doses of gamma-irradiation of mammalian cells.

The methodology previously described for measuring mutagenesis has been applied to the study of mutation prevention in immortalized G2 phase human lymphocytes exposed to 25 and 50 cGy of gamma-radiation. Caffeine prevents repair of mutations. Two times 10(-4) M beta-estradiol applied for 2.5 h markedly decreases induced mutations and affects male and female cells similarly. Quantitative measurement of mutagenesis in cells of different individuals and the effect of various agents on mutation yield should be important in prevention of cancer and other mutational disease.

Caffeine↗
PubMed1999

A measure of uncertainty: the nature of vulnerability and its relationship to malnutrition.

Terms such as 'vulnerability' and 'insecurity' are used widely in the general nutrition literature as well as in work on humanitarian response. Yet these words are used rather loosely. This paper argues that more clarity in their usage would benefit those seeking a bridge between development and humanitarian problems. Since vulnerability is not fully coincident with malnutrition, poverty or other conventional indices of human deprivation, public action must be based on a better understanding of the nature of crises and human uncertainty beyond physiological and nutritional outcomes. More attention is needed to be paid to the context-specific nature of risks, the capacity of households to manage such risks and the potential for public action to bolster indigenous capacity through targeted development investments, not just relief.

Altruism↗
PubMed1999

Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins.

Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

Animals↗
PubMed1999

The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions.

Estrogen receptors (ERs alpha and beta) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ERalpha AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ERalpha utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ERalpha AF-1 and weakly inhibited by AF-2. Indeed, ERalpha truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ERbeta, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors.

Binding Sites↗
PubMed1999

Titration by estrogen receptor activation function-2 of targets that are downstream from coactivators.

Cross-interference (squelching) among nuclear receptors has been proposed to reflect the titration of coactivators that bind the receptors in a hormone-dependent manner. We have tested whether the coactivators are the only target titrated during squelching of one receptor by another, or whether proteins needed for coactivator function are titrated as well. That the coactivators are indeed one target of squelching is apparent. The isolated ligand-binding domain of the estrogen receptor (ER-LBD) squelches transcriptional activation by the thyroid hormone receptor (TR) only when the LBD is bound to ligands that promote coactivator interactions and only when regions of the LBD that promote coactivator interactions are undisturbed. Furthermore, the ER-LBD and the TR compete in vitro for the related p160 coactivators, SRC1a and GRIP1 (glucocorticoid receptor interacting protein 1), or the putative corepressor, RIP140. Finally TR action becomes more potent when coactivator levels are raised. Nonetheless, supplying excess SRC1a or GRIP1 does not abolish squelching by the ER. In fact, squelching becomes even more severe when coactivators are abundant. Supplying combinations of coactivators from the p160 class and the CREB-binding protein (CBP)/p300 class makes squelching most severe. Elevated RIP140 inhibits TR action, but also protects the residual TR action from squelching by the ER-LBD. We conclude that ER-LBD squelches TR both by titrating p160-CBP coactivators and additionally by cooperating with the coactivators to titrate a second factor. The second factor would be needed by the TR for coactivator-mediated transcriptional stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Mutation measurement in mammalian cells. IV: Comparison of gamma-ray and chemical mutagenesis.

The interaction of chemical mutagens with mammalian cells is much more complex than that of gamma-irradiation because of the different ways in which chemical agents react with cell and medium components. Nevertheless, the system previously described for analysis of mutagenesis by gamma-radiation appears applicable to chemical mutagenesis. The approach involves measurement of cell survival, use of caffeine to inhibit repair, analysis of mitotic index changes, and quantitation of microscopically visible structural changes in mitotic chromosomes. The behavior of a variety of chemical mutagens and nonmutagens in this system is described and compared with that of gamma-irradiation. The procedure is simple and the results reasonably quantitative though less so than those of gamma-irradiation. The procedure can be used for environmental monitoring, analysis of mutational events, and individual and epidemiological testing. Mutational events should be classified as primary or secondary depending on whether they represent initial genomic insult, or genomic changes resulting from primary mutation followed by structural changes due to metabolic actions. While caffeine has multiple effects on the mammalian genome, when used under the conditions specified here it appears to act principally as an inhibitor of mutation repair, and so affords a measure of the role of repair in the action of different mutagens on cells in the G2 phase of the life cycle.

Animals↗
PubMed1998

Genome exposure and regulation in mammalian cells.

A method of measurement of exposed DNA (i.e. hypersensitive to DNase I hydrolysis) as opposed to sequestered (hydrolysis resistant) DNA in isolated nuclei of mammalian cells is described. While cell cultures exhibit some differences in behavior from day to day, the general pattern of exposed and sequestered DNA is satisfactorily reproducible and agrees with results previously obtained by other methods. The general pattern of DNA hydrolysis exhibited by all cells tested consists of a curve which at first rises sharply with increasing DNase I, and then becomes almost horizontal, indicating that roughly about half of the nuclear DNA is highly sequestered. In 4 cases where transformed cells (Raszip6, CHO, HL60 and PC12) were compared, each with its more normal homolog (3T3, and the reverse transformed versions of CHO, HL60 and PC12, achieved by dibutyryl cyclic AMP [DBcAMP], retinoic acid, and nerve growth factor [NGF] respectively), the transformed form displayed less genome exposure than the nontransformed form at every DNase I dose tested. When Ca++ was excluded from the hydrolysis medium in both the Raszip6-3T3 and the CHO-DBcAMP systems, the normal cell forms lost their increased exposure reverting to that of the transformed forms. Therefore Ca++ appears necessary for maintenance of the DNA in the more highly exposed state characteristic of the nontransformed phenotype. LiCl increases the DNA exposure of all transformed cells tested. Dextran sulfate and heparin each can increase the DNA exposure of several different cancers. Colcemid prevents the increase of exposure of CHO by DBcAMP but it must be administered before or simultaneously with the latter compound. Measurements on mouse biopsies reveal large differences in exposure in different normal tissues. Thus, the exposure from adult liver cells was greater than that of adult brain, but both fetal liver and fetal brain had significantly greater exposure than their adult counterparts. Exposure in normal human fibroblasts as revealed by in situ nick translation reveals a nuclear distribution pattern around the periphery, around the nucleoli and in punctate positions in the nuclear interior in parts of both S and G1 phases of the cell cycle. The same exposure pattern is duplicated by the pattern of DNA synthesis in S cells. It would appear that these nuclear regions represent positions of special activity. The previously proposed theory of genome regulation in mammalian cells is supported by these findings. The theory proposes that: a) gene activity requires exposure of the given locus followed by action of transcription factors on the exposed genes; b) the fiber system of the cell (cytoskeleton, nuclear fibers, and extracellular fibers) are required for normal exposure; c) active sites for gene expression and replication consist of the nuclear periphery where differentiation genes particularly are exposed; the nucleoli where at least some housekeeping genes are exposed; and possibly also punctate regions in the interior; d) noncoding sequences play a critical role in genome regulation, possibly including the transport of loci to be activated to appropriate exposure transcriptional and replicating locations. Cancer cells have lost specific differentiation gene activities, at least sometimes because of mutation of appropriate exposure genes; at least some protooncogenes and tumor suppressor genes are responsible for exposure and transport of specific differentiation gene loci to their appropriate exposure sites in the nucleus and for inducing exposure.

3T3 Cells↗
PubMed1998

Molecular and structural biology of thyroid hormone receptors.

1. Thyroid hormone receptors (TR) are expressed from two separate genes (alpha and beta) and belong to the nuclear receptor superfamily, which also contains receptors for steroids, vitamins and prostaglandins. 2. Unliganded TR are bound to DNA thyroid hormone response elements (TRE) predominantly as homodimers, or as heterodimers with retinoid X-receptors (RXR), and are associated with a complex of proteins containing corepressor proteins. Ligand binding promotes corepressor dissociation and binding of a coactivator. 3. Recent studies from our group have focused on the acquisition and use of X-ray crystallographic structures of ligand-binding domains (LBD) of both the rat (r) TR alpha and the human (h) TR beta bound to several different ligands. We have also developed ligands that bind selectively to the TR beta, which may provide ways to explore the differential functions of TR alpha compared with TR beta isoforms. 4. The LBD is comprised mostly of alpha-helices. The ligand is completely buried in the receptor and forms part of its hydrophobic core. Kinetic studies suggest that the limiting step in formation of high-affinity ligand-receptor complexes is the rate of folding of the receptor around the ligand. Ligands can be fitted tightly in the ligand-binding pocket and small differences in this fitting may explain many structure-activity relationships. Interestingly, analysis of the structures of antagonists suggests that they have chemical groups, 'extensions', that could impair receptor folding around them and, thus, prevent the agonist-induced conformation changes in the receptor. 5. The TR structures allowed us to see that the mutations that occur in the syndrome of generalized resistance to thyroid hormone are located in the vicinity of the ligand-binding pocket. 6. X-ray structure of the TR has also been used to guide construction of mutations in the TR surface that block binding of various proteins important for receptor function. Studies with these TR mutants reveal that the interfaces for homo- and heterodimerization map to similar residues in helix 10 and 11 and also allow the definition of the surface for binding of coactivators, which appears to be general for nuclear receptors. Formation of this surface, which involves packing of helix 12 of the TR into a scaffold formed by helices 3 and 5, appears to be the major change in the receptor structure induced by hormone occupancy.

Animals↗
PubMed1998

Estrogen receptor activation function 1 works by binding p160 coactivator proteins.

Estrogen receptor-alpha contains two transactivation functions, a weak constitutive activation function (AF-1) and a hormone-dependent activation function (AF-2). AF-2 works by recruiting a large coactivator complex, composed of one or more p160s, CREB-binding protein (CBP)/p300, and P/CAF (p300 and CBP-associated factor), via direct contacts with the p160s. We report here that independent AF-1 activity also requires p160 contacts. Unlike AF-2, which binds signature NR boxes in the center of the p160 molecule, AF-1 binds to sequences near the p160 C terminus. We propose that the ability of AF-1 and AF-2 to interact with separate surfaces of the same coactivator is important for the ability of these transactivation functions to synergize.

Acetyltransferases↗
PubMed1998

Synthetic porous ceramic compared with autograft in scoliosis surgery. A prospective, randomized study of 341 patients.

We have evaluated the use of a synthetic porous ceramic (Triosite) as a substitute for bone graft in posterior spinal fusion for idiopathic scoliosis. In a prospective, randomised study 341 patients at five hospitals in the UK and France were randomly allocated either to autograft from the iliac crest or rib segments (171) or to receive Triosite blocks (170). All patients were assessed after operation and at 3, 6, 12 and 18 months. The two groups were similar with regard to all demographic and baseline variables, but the 184 treated in France (54%) had Cotrel-Dubouset instrumentation and the 157 treated in the UK usually had Harrington-Luque implants. In the Triosite group the average Cobb angle of the upper curve was 56 degrees, corrected to 24 degrees (57%). At 18 months, the average was 26 degrees (3% loss). In the autograft group the average preoperative upper curve of 53 degrees was corrected to 21 degrees (60%). At 18 months the mean curve was 25 degrees (8% loss). Pain levels after operation were similar in the two groups, being mild in most cases. In the Triosite group only three patients had problems of wound healing, but in the autograft group, 14 patients had delayed healing, infection or haematoma in the spinal wound. In addition, 15 autograft patients had pain at the donor site at three months. Seven had infections, two had haematoma and four had delayed healing. The haematological and serum biochemistry results showed no abnormal trends and no significant differences between the groups. There were no adverse events related to the graft material and no evidence of allergenicity. Our results suggest that Triosite synthetic porous ceramic is a safe and effective substitute for autograft in these patients. Histological findings on biopsy indicate that Triosite provides a favourable scaffolding for the formation of new bone and is gradually incorporated into the fusion mass.

Adolescent↗
PubMed1998

Vernix caseosa peritonitis as a rare complication of cesarean section. A case report.

BACKGROUND: Vernix caseosa peritonitis is a rare complication of cesarean section with distinctive histopathologic findings. CASE: Vernix caseosa peritonitis occurred in a 28-year-old, pregnant woman following emergency cesarean section. Exploratory laparotomy revealed right subphrenic and perihepatic fluid collections with multiple small abdominal abscesses. Cytopathologic examination of the peritoneal lavage specimen showed a purulent inflammatory exudate admixed with occasional squamous debris, portions of hair shafts, meconium pigment, and foamy and multinucleated giant cells. During hospitalization the patient had developed Clostridium perfringens acute endometritis, presumably due to aggressive treatment with broad-spectrum antibiotics. CONCLUSION: Pathologists are in a unique position to make the correct diagnosis in an otherwise-ambiguous clinical presentation because of the distinctive histopathologic and cytopathologic findings of this entity. Computed tomography-guided fine needle aspiration cytologic evaluation can be diagnostic in such cases.

Adult↗
PubMed1997

Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites.

The transactivation properties of the two estrogen receptors, ERalpha and ERbeta, were examined with different ligands in the context of an estrogen response element and an AP1 element. ERalpha and ERbeta were shown to signal in opposite ways when complexed with the natural hormone estradiol from an AP1 site: with ERalpha, 17beta-estradiol activated transcription, whereas with ERbeta, 17beta-estradiol inhibited transcription. Moreover, the antiestrogens tamoxifen, raloxifene, and Imperial Chemical Industries 164384 were potent transcriptional activators with ERbeta at an AP1 site. Thus, the two ERs signal in different ways depending on ligand and response element. This suggests that ERalpha and ERbeta may play different roles in gene regulation.

Animals↗