Effects of p53 mutations on apoptosis in mouse intestinal and human colonic adenomas

We have examined the effects of inactivation of the p53 tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the intestine: in early adenomas where p53 mutations are rare and in highly dysplastic adenomas where loss of p53 occurs frequently. Homozygosity for an inactivating germ-line mutation of p53 had no effect on the incidence or the rate of progression of ApcMin/+-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the effect of p53 loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during colon tumor progression with loss of heterozygosity at the p53 locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that p53 is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type p53 retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of apoptosis.

Transforming growth factor β-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells

Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor β (TGF-β)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-β signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-β receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-β treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-β-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-β in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-β signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-β receptor complex is an essential step in signal transduction by TGF-β for both inhibition of cell proliferation and activation of the PAI-1 promoter.

Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G1/S checkpoint and maintain high levels of p27KIP1 mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27KIP1 is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin α2β1, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.

IL-7 is critical for homeostatic proliferation and survival of naïve T cells

In T cell-deficient conditions, naïve T cells undergo spontaneous “homeostatic” proliferation in response to contact with self-MHC/peptide ligands. With the aid of an in vitro system, we show here that homeostatic proliferation is also cytokine-dependent. The cytokines IL-4, IL-7, and IL-15 enhanced homeostatic proliferation of naïve T cells in vitro. Of these cytokines, only IL-7 was found to be critical; thus, naïve T cells underwent homeostatic proliferation in IL-4− and IL-15− hosts but proliferated minimally in IL-7− hosts. In addition to homeostatic proliferation, the prolonged survival of naïve T cells requires IL-7. Thus, naïve T cells disappeared gradually over a 1-month period upon adoptive transfer into IL-7− hosts. These findings indicate that naïve T cells depend on IL-7 for survival and homeostatic proliferation.

Molecular markers reveal that population structure of the human pathogen Candida albicans exhibits both clonality and recombination.

The life history of Candida albicans presents an enigma: this species is thought to be exclusively asexual, yet strains show extensive phenotypic variation. To address the population genetics of C. albicans, we developed a genetic typing method for codominant single-locus markers by screening randomly amplified DNA for single-strand conformation polymorphisms. DNA fragments amplified by arbitrary primers were initially screened for single-strand conformation polymorphisms and later sequenced using locus-specific primers. A total of 12 single base mutations and insertions were detected from six out of eight PCR fragments. Patterns of sequence-level polymorphism observed for individual strains detected considerable heterozygosity at the DNA sequence level, supporting the view that most C. albicans strains are diploid. Population genetic analyses of 52 natural isolates from Duke University Medical Center provide evidence for both clonality and recombination in C. albicans. Evidence for clonality is supported by the presence of several overrepresented genotypes, as well as by deviation of genotypic frequencies from random (Hardy-Weinberg) expectations. However, tests for nonrandom association of alleles across loci reveal less evidence for linkage disequilibrium than expected for strictly clonal populations. Although C. albicans populations are primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

Increased histone H1 phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts.

Fibroblasts derived from embryos homozygous for a disruption of the retinoblastoma gene (Rb) exhibit a shorter G1 than their wild-type counterparts, apparently due to highly elevated levels of cyclin E protein and deregulated cyclin-dependent kinase 2 (CDK2) activity. Here we demonstrate that the Rb-/- fibroblasts display higher levels of phosphorylated H1 throughout G1 with the maximum being 10-fold higher than that of the Rb+/+ fibroblasts. This profile of intracellular H1 phosphorylation corresponds with deregulated CDK2 activity observed in in vitro assays, suggesting that CDK2 may be directly responsible for the in vivo phosphorylation of H1. H1 phosphorylation has been proposed to lead to a relaxation of chromatin structure due to a decreased affinity of this protein for chromatin after phosphorylation. In accord with this, chromatin from the Rb-/- cells is more susceptible to micrococcal nuclease digestion than that from Rb+/+ fibroblasts. Increased H1 phosphorylation and relaxed chromatin structure have also been observed in cells expressing several oncogenes, suggesting a common mechanism in oncogene and tumor suppressor gene function.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Growth suppression by p16ink4 requires functional retinoblastoma protein.

p16ink4 has been implicated as a tumor suppressor that is lost from a variety of human tumors and human cell lines. p16ink4 specifically binds and inhibits the cyclin-dependent kinases 4 and 6. In vitro, these kinases can phosphorylate the product of the retinoblastoma tumor suppressor gene. Thus, p16ink4 could exert its function as tumor suppressor through inhibition of phosphorylation and functional inactivation of the retinoblastoma protein. Here we show that overexpression of p16ink4 in certain cell types will lead to an arrest in the G1 phase of the cell cycle. In addition, we show that p16ink4 can only suppress the growth of human cells that contain functional pRB. Moreover, we have compared the effect of p16ink4 expression on embryo fibroblasts from wild-type and RB homozygous mutant mice. Wild-type embryo fibroblasts are inhibited by p16ink4, whereas the RB nullizygous fibroblasts are not. These data not only show that the presence of pRB is crucial for growth suppression by p16ink4 but also indicate that the pRB is the critical target acted upon by cyclin D-dependent kinases in the G1 phase of the cell cycle.

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.

Post-Conflict Maternal Communication, Children's Understandings, and Child Functioning in Violent and Non-Violent Families

The last two decades have been marked by a growing public awareness of family violence. Research by social scientists has suggested that family violence is widespread (Gelles and Straus, 1988). It is estimated that every year 1.8 to 4 million women are physically abused by their partners (Novello, 1992). In fact, more women are abused by their husbands or boyfriends than are injured in car accidents, muggings, or rapes (Jaffe, Wolfe, and Wilson, 1990). A recent prevalence study by Fantuzzo, Boruch, Beriama, Atkins, and Marcus (1997) found that children were disproportionately present in households where there was a substantial incident of adult female assault. Experts estimate that 3.3 to 10 million children are exposed to marital violence each year (Carlson, 1984; Straus, 1991). Until recently, most researchers did not consider the impact of parental conflict on the children who witness this violence. The early literature in this field primarily focused on the incidence of violence against women and the inadequate response of community agencies (Jaffe et al, 1990). The needs of children were rarely considered. However, researchers have become increasingly aware that children exposed to marital violence are victims of a range of psychological maltreatment (e.g., terrorizing, isolation;Hart, Brassared & Karlson, 1996) and are at serious risk for the development of psychological problems (Fantuzzo, DePaola, Lambert, Martino, Anderson, and Sutton, 1991). Jouriles, Murphy and O'Leary (1989) found that children of battered women were four times more likely to exhibit psychopathology as were children living in non-violent homes. Further, researchers have found associations between childhood exposure to parental violence and the expression of violence in adulthood (Carlson, 1990). Existing research suggests that children who have witnessed marital violence manifest numerous emotional, social, and behavioral problems (Sternberg et al., 1993; Fantuzzo et al., 1991; Jaffe et al, 1990). Studies have found that children of battered women exhibit more internalizing and externalizing behavior problems than non-witnesschildren (Hughes and Fantuzzo, 1994; McCloskey, Figueredo, and Koss, 1995). In addition, children exposed to marital violence have been found to exhibit difficulties with social problem-solving, and have lower levels of social competence than nonwitnesses (Rosenberg, 1987; Moore, Pepler, Weinberg, Hammond, Waddell, & Weiser, 1990). Other reported difficulties include low self esteem (Hughes, 1988), poor school performance (Moore et al., 1990) and problems with aggression (Holden & Ritchie, 1991; Jaffe, Wolfe, Wilson, & Zak, 1986). Further, within the last decade, researchers have found that some children are traumatized by the witnessing experience, showing elevated levels of posttraumatic stress symptoms (Devoe & Graham-Bermann, 1997; Rossman, Bingham, & Emde, 1996; Kilpatrick, Litt, & Williams, 1997). These findings corroborate clinical reports that describe many exposed children as experiencing trauma reactions. It appears that the negative effects of witnessing marital violence are numerous and varied, ranging from mild emotional and behavioral problems to clinically significant levels of posttraumatic stress symptoms. These incidence figures and research findings indicate that children's exposure to violence is a significant problem in our nation today and has serious implications for the future.

Radiomic Texture Analysis Mapping Predicts Areas of True Functional MRI Activity.

Individual analysis of functional Magnetic Resonance Imaging (fMRI) scans requires user-adjustment of the statistical threshold in order to maximize true functional activity and eliminate false positives. In this study, we propose a novel technique that uses radiomic texture analysis (TA) features associated with heterogeneity to predict areas of true functional activity. Scans of 15 right-handed healthy volunteers were analyzed using SPM8. The resulting functional maps were thresholded to optimize visualization of language areas, resulting in 116 regions of interests (ROIs). A board-certified neuroradiologist classified different ROIs into Expected (E) and Non-Expected (NE) based on their anatomical locations. TA was performed using the mean Echo-Planner Imaging (EPI) volume, and 20 rotation-invariant texture features were obtained for each ROI. Using forward stepwise logistic regression, we built a predictive model that discriminated between E and NE areas of functional activity, with a cross-validation AUC and success rate of 79.84% and 80.19% respectively (specificity/sensitivity of 78.34%/82.61%). This study found that radiomic TA of fMRI scans may allow for determination of areas of true functional activity, and thus eliminate clinician bias.

Todo o poder às redes

O sociólogo que comanda um dos grandes centros de análise de big data no Brasil diz que os políticos só vão recuperar legitimidade quando aprenderem que "curtir" é coisa séria para detectar tendências e medir o pulso das aspirações sociais ganhou volume e tempo real no oceano de informações do big data, nome que se dá à gigantesca quantidade de dados produzidos diariamente na internet. É nessa mina inesgotável que o sociólogo carioca Marco Aurelio Ruediger, da Fundação Getulio Vargas, abastece a Diretoria de Análise de Políticas Públicas, um centro de estudo da visão que os brasileiros têm da máquina estatal e dos poderes da República.

Minority students : a research appraisal /

Includes indexes.