Recent outbreaks of infectious syphilis, United Kingdom, January 2012 to April 2014.

Six outbreaks of infectious syphilis in the United Kingdom, ongoing since 2012, have been investigated among men who have sex with men (MSM) and heterosexual men and women aged under 25 years. Interventions included case finding and raising awareness among healthcare professionals and the public. Targeting at-risk populations was complicated as many sexual encounters involved anonymous partners. Outbreaks among MSM were influenced by the use of geospatial real-time networking applications that allow users to locate other MSM within close proximity.

Multicentre review of lymph node harvest in colorectal cancer: are we understaging colorectal cancer patients?

BACKGROUND: Lymph node examination in colorectal cancer is of vital importance for accurate staging. Patients who have fewer nodes examined may be understaged and not offered adjuvant chemotherapy. The national institute of clinical excellence and the association of coloproctology of Great Britain and Ireland both recommend that 12 nodes should be examined for accurate staging. The aim of this study was to assess lymph node harvest at five hospitals in the northwest of England in respect to these guidelines. MATERIALS AND METHODS: This study is a retrospective review of all colorectal cancer resections over a 1-year period at five hospitals. RESULTS: Two hospitals met the national guidelines of a median of 12 or more nodes. Overall, over 50% of colorectal cancers contained fewer than 12 nodes. Fifty-three point seven percent (53.7%) of Dukes B patients did not have 12 nodes in their specimens and may therefore be understaged. There was a significant variation between hospitals in terms of the number of cancers with 12 or more nodes (P < 0.0001) and the number of Dukes B cancers with 12 or more nodes (P < 0.008). CONCLUSION: Over 50% of all colorectal cancer specimens contain fewer than 12 lymph nodes despite clear national guidelines. This is of particular importance to Dukes B cancers where over 53% of cases may be understaged and not offered adjuvant therapy. Significant variation exists between hospitals within the same region.

Biological significance of a small highly conserved region in the N terminus of the p53 tumour suppressor protein.

The p53 tumour suppressor protein plays a central role in maintaining genomic integrity in eukaryotic cells. The most significant biological function of p53 is to act as a sequence-specific DNA-binding transcription factor, which can induce the expression of a variety of target genes in response to diverse stress stimuli. The p53 protein contains six highly conserved regions, one of which, termed Box I, is located in the N-terminal transactivation domain (amino acid residues 13 and 26). The second half of the Box I region is crucial for the interaction with the basal transcription machinery and is thus required for p53's activity as a transcription factor. The same region also binds to Mdm2. Since p53 is targeted by Mdm2 for ubiquitin-mediated proteasome-dependent degradation, this region is also essential for the regulation of p53's stability in response to stress signals. Although the first half of Box I is highly conserved, its biological function is not clearly defined. The aim of this study was to characterise this conserved region and investigate its role in the biological functions of p53. We have generated short deletions and point mutations within this region and analysed their effect on p53 function and regulation. Biochemical analyses demonstrate that deletion of residues 13 to 16 significantly increases both the transcriptional transactivation and G(2) arrest-inducing activities of murine p53. Residues 13 to 16 appear to function as a regulatory element in p53, modulating p53-dependent transcriptional transactivation and cell-cycle arrest, possibly by affecting the structural stability of the core domain of the protein. In support of this, the deletion was found to induce second-site reversion of the Val135 temperature-sensitive mutant of murine p53.

An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo.

The p53 tumour suppressor protein is down-regulated by the action of Mdm2, which targets p53 for rapid degradation by the ubiquitin-proteasome pathway. The p14ARF protein is also a potent tumour suppressor that acts by binding to Mdm2 and blocking Mdm2-dependent p53 degradation and transcriptional silencing. We have screened a series of overlapping synthetic peptides derived from the p14ARF protein sequence and found that a peptide corresponding to the first 20 amino acids of ARF (Peptide 3) could bind human Mdm2. The binding site for Peptide 3 on Mdm2 was determined by deletion mapping and lies adjacent to the binding site of the anti-Mdm2 antibody 2A10, which on microinjection into cells can activate p53-dependent transactivation of a reporter plasmid. To determine whether Peptide 3 could similarly activate p53, we expressed a fusion of green fluorescent protein and Peptide 3 in MCF7 and U-2 OS cells and were able to demonstrate induction of p53 protein and p53-dependent transcription. Peptide 3 was able to block in vitro ubiquitination of p53 mediated by Mdm2. Small peptides which are sufficient to block degradation of p53 could provide therapeutic agents able to restore p53-dependent cell death pathways in tumours that retain wild-type p53 expression.

Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA.

Biochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full-length mdm2 does not bind stably to p53-DNA complexes, contrasting with C-terminal truncations of mdm2 which do bind stably to p53-DNA complexes. In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX-I domain of p53 becomes concealed when either p53 binds to DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C-terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full-length mdm2 with p53 lacking the BOX-I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX-I domain, addition of RNA activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53-mdm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein; (2) the C-terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA.

Effects on normal fibroblasts and neuroblastoma cells of the activation of the p53 response by the nuclear export inhibitor leptomycin B.

p53 tumour suppressor protein levels and p53-dependent transcriptional activity have been recently shown to increase in cells treated with leptomycin B (LMB), an inhibitor of nuclear export. Experiments presented here show that LMB treatment leads to growth arrest and a senescence-like phenotype in human normal fibroblast cultures. This effect is reversible after removal of the drug and further passage by trypsinization. Instead, LMB has a strong cytotoxic effect on human neuroblastoma cell lines even at nanomolar concentrations. In both these cell types the effects of LMB are attenuated when the activity of the endogenous wild type p53 protein is abrogated by overexpression of a dominant negative p53 mutant. We conclude that the induction of the p53 response by LMB plays an important role in the effects of this drug on cultured cells.

SUMO-1 modification activates the transcriptional response of p53.

The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In normal cells p53 is constitutively ubiquitylated by the Mdm2 ubiquitin ligase. When the p53 response is activated by stress signals p53 levels rise due to inhibition of this degradative pathway. Here we show that p53 is modified by the small ubiquitin-like protein SUMO-1 at a single site, K386, in the C-terminus of the protein. Modification in vitro requires only SUMO-1, the SUMO-1 activating enzyme and ubc9. SUMO-1 and ubiquitin modification do not compete for the same lysine acceptor sites in p53. Overexpression of SUMO-1 activates the transcriptional activity of wild-type p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. The SUMO-1 modification pathway therefore acts as a potential regulator of the p53 response and may represent a novel target for the development of therapeutically useful modulators of the p53 response.

p53 suppresses the activation of the Bcl-2 promoter by the Brn-3a POU family transcription factor.

The Brn-3a POU family transcription factor has been shown to strongly activate expression of the Bcl-2 proto-oncogene and thereby protect neuronal cells from programmed cell death (apoptosis). This activation of the Bcl-2 promoter by Brn-3a is strongly inhibited by the p53 anti-oncogene protein. This inhibitory effect of p53 on Brn-3a-mediated transactivation is observed with nonoverlapping gene fragments containing either the Bcl-2 p1 or p2 promoters but is not observed with other Brn-3a-activated promoters such as in the gene encoding alpha-internexin or with an isolated Brn-3a binding site from the Bcl-2 promoter linked to a heterologous promoter. In contrast, p53 mutants, which are incapable of binding to DNA, do not affect Brn-3a-mediated activation of the Bcl-2 p1 and p2 promoters. Moreover, Brn-3a and p53 have been shown to bind to adjacent sites in the p2 promoter and to directly interact with one another, both in vitro and in vivo, with this interaction being mediated by the POU domain of Brn-3a and the DNA binding domain of p53. The significance of these effects is discussed in terms of the antagonistic effects of Bcl-2 and p53 on the rate of apoptosis and the overexpression of Brn-3a in specific tumor cell types.

An inhibitor of nuclear export activates the p53 response and induces the localization of HDM2 and p53 to U1A-positive nuclear bodies associated with the PODs.

Leptomycin B is a cytotoxin which directly interacts with and inhibits the action of CRM1, an essential mediator of the nuclear exit of proteins containing nuclear export signals (NES) of the HIV1 REV type. We show that addition of leptomycin B to human primary fibroblasts increased the levels of the p53 tumor suppressor protein. This was accompanied by the induction of p53-dependent transcriptional activity in cultured cells and an increase in the levels of the products of two p53-responsive genes, the p21(CIP1/WAF1) and HDM2 proteins. Leptomycin B induced the accumulation of p53 and HDM2 in the nucleus and the appearance of discrete nuclear aggregates containing both proteins. It has been reported that the transcriptional activity of p53 is modulated by its interaction with the HDM2 protein which also targets p53 for rapid degradation. Using a model cell line conditionally expressing MDM2, the murine analogue of HDM2, we present evidence indicating that leptomycin B abrogates MDM2's role in p53 degradation and that the accumulation of p53 in distinct nuclear bodies is mediated by MDM2. Since HDM2 has recently been shown to contain a functional NES of the REV type, the most likely explanation for our results is that the effect of leptomycin B on HDM2 and p53 is due to the inhibition of nuclear export. The ability to visualize sites where p53 and HDM2 colocalize provides a new approach to study the association between the two proteins in vivo. These p53/HDM2-positive nuclear foci were found to also contain the U1A snRNP A and to be juxtaposed to the PML oncogenic domains.

A resistant genetic background leading to incomplete penetrance of intestinal neoplasia and reduced loss of heterozygosity in ApcMin/+ mice.

Previous studies of Min/+ (multiple intestinal neoplasia) mice on a sensitive genetic background, C57BL/6 (B6), showed that adenomas have lost heterozygosity for the germ-line ApcMin mutation in the Apc (adenomatous polyposis coli) gene. We now report that on a strongly resistant genetic background, AKR/J (AKR), Min-induced adenoma multiplicity is reduced by about two orders of magnitude compared with that observed on the B6 background. Somatic treatment with a strong mutagen increases tumor number in AKR Min/+ mice in an age-dependent manner, similar to results previously reported for B6 Min/+ mice. Immunohistochemical analyses indicate that Apc expression is suppressed in all intestinal tumors from both untreated and treated AKR Min/+ mice. However, the mechanism of Apc inactivation in AKR Min/+ mice often differs from that observed for B6 Min/+ mice. Although loss of heterozygosity is observed in some tumors, a significant percentage of tumors showed neither loss of heterozygosity nor Apc truncation mutations. These results extend our understanding of the effects of genetic background on Min-induced tumorigenesis in several ways. First, the AKR strain carries modifiers of Min in addition to Mom1. This combination of AKR modifiers can almost completely suppress spontaneous intestinal tumorigenesis associated with the Min mutation. Second, even on such a highly resistant genetic background, tumor formation continues to involve an absence of Apc function. The means by which Apc function is inactivated is affected by genetic background. Possible scenarios are discussed.

The Development and Validation of Scales Assessing Students' Achievement Goal Orientations

Achievement goal theory has emerged as a major new direction in motivational research. A distinction is made among conceptually different achievement goal orientations including the goal to develop ability (task goal orientation), the goal to demonstrate ability (ability-approach goal orientation), and the goal to avoid the demonstration of lack of ability (ability-avoid goal orientation). Scales assessing each of these goal orientations were developed over an eight year period by a group of researchers at the University of Michigan. The results of studies conducted with seven different samples of elementary and middle school students are used to describe the internal consistency, stability, and construct validity of the scales. Comparisons of these scales with those developed by Nicholls and his colleagues provide evidence of convergent validity. Confirmatory factor analysis attests to the discriminant validity of the scales. Copyright 1998 Academic Press.

The N terminus of the murine p53 tumour suppressor is an independent regulatory domain affecting activation and thermostability.

The contribution of each of the structural domains of p53 to its function has been discussed widely in the literature. Crystallographic studies have revealed much about the structure of the core DNA binding domain, but as it has not been possible to use this approach for the intact protein, the effect of the domains flanking the core must be investigated by more indirect techniques. In this study a series of truncated murine p53 proteins has been investigated for DNA binding activity at 4 degrees C and 37 degrees C, transcriptional activation, and tumour suppression activity. Full-length p53, and truncations lacking the N terminus, purified from a baculovirus expression system all show latency for DNA binding; that is, they must be activated to bind by association with a C-terminal antibody such as PAb421. This demonstrates that latency for DNA binding is independent of the N terminus. Truncations lacking the C-terminal oligomerisation domain, and the isolated core domain, can only be activated to bind DNA and PAb1620 (an antibody recognising the wild-type conformation of the core domain) in the presence of cross-linking antibodies, while murine core only binds to DNA in the presence of PAb1620. An analysis of the thermostability of DNA binding revealed that antibodies that bind the N terminus of p53 could protect the protein against loss of activity at 37 degrees C. C-terminal antibodies, however, were ineffective unless the N-terminal 37 amino acid residues were absent. The N terminus may retain some secondary structure, since it is the main contributor to the anomalous migration in SDS-polyacrylamide gels. Our results suggest that the N terminus has a destabilising effect that influences conformation of p53 at 37 degrees C, so cellular proteins binding to the N terminus in vivo may modulate p53 conformation and stability. The effects on thermostability are also direct evidence showing that antibodies binding to N-terminal deletions create a conformational change in the rest of the molecule. In addition, longer deletions of the C terminus reduce the ability of p53 to transactivate target genes and inactivate tumour suppression activity, while truncations of the N terminus retain partial tumour suppression activity. Our results clearly show participation of both the N and C termini in the regulation of all the functions of p53 at 37 degrees C, indicating that distinct, independent domains interact with each other within, the flexible structure of p53.

The Effect of Achievement Goals: Does Level of Perceived Academic Competence Make a Difference?

Researchers using a goal orientation framework have hypothesized that learning goals are associated with adaptive patterns of behavior, regardless of the level of perceived ability. In contrast, perceived ability is hypothesized to moderate the relation between performance goals and patterns of adaptive or maladaptive behavior. We examined this hypothesis in two samples of seventh grade middle school students, focusing on the math domain in one sample and on the English domain in the other. Using two different statistical methods, median split and multiple regression, we found only little support for the role of perceived competence as a moderator between performance goals and patterns of behavior. Contrary to what has been suggested, we found some evidence that perceived competence moderated the relation between learning goals and behavior. Implications of these findings for recent efforts to use goal theory to reform classrooms and schools are discussed. Copyright 1997Academic Press

p53 protein stability in tumour cells is not determined by mutation but is dependent on Mdm2 binding.

The tumour suppressor protein p53 is expressed at very low levels in normal cells but accumulates in response to DNA damaging agents such as u.v. irradiation. This increase is accompanied by transcriptional upregulation of the expression of a number of proteins including Mdm2 which can in turn inhibit p53 dependent transcriptional activation, creating a feedback loop resulting in down-regulation of p53 activity. Mutant p53 proteins are however frequently detected at constitutively high levels in many tumours and tumour cell lines, indeed this phenomenon has been used in several studies to diagnose p53 mutation in patient tumours. We show here that expression of mouse mutant p53 in tumour cell lines of this type results in high levels of both the endogenous p53 protein and the exogenously expressed mutant mouse protein, whereas the human tumour line MCF7 does not exhibit high levels of either endogenous human or exogenously expressed mouse mutant p53 unless stabilisation is induced by DNA damage. This suggests that the stability of mutant p53 is not intrinsic to mutant p53 protein structure but may vary in different cell backgrounds. We present evidence that p53 protein stability in tumour cell lines is determined by association with the Mdm2 tumour suppressor protein, and that p53 mutants which are unable to bind Mdm2 are stable in MCF7 cells. We propose that tumour lines which express high levels of transcriptionally inactive mutant p53 are unable to induce the expression of the Mdm2 protein which would normally provide a feedback mechanism down-regulating p53 protein levels in the absence of DNA damage signals. MCF7 cells however express a transcriptionally active p53 and retain the feedback regulation of p53 protein levels by Mdm2.

Changes in Achievement Goal Orientations, Perceived Academic Competence, and Grades across the Transition to Middle-Level Schools

Goal orientation theory was used to examine changes in student motivation during the transition from elementary to middle school. Surveys were given to 341 students in the fifth grade in elementary and again in sixth grade in middle school. Students were more oriented to task goals (wanting to improve their competency), perceived a greater emphasis on task goals during instruction, and felt more academically competent in fifth grade in elementary school than in sixth grade in middle school. They perceived a greater emphasis on performance goals (an emphasis on relative ability and right answers) in middle school than in elementary school. Several interactions emerged between year (fifth grade, sixth grade), and both student level of ability (higher, lower, based on standardized achievement tests) and subject domain (math, English).

APC expression in normal human tissues.

The tumour suppressor gene APC codes for a 2843-amino acid protein whose precise functions are still poorly understood. This paper describes the development of two new antisera to APC (to amino- and carboxy-terminal epitopes) which permit localization of the protein by immunohistochemistry in archival paraffin sections. The protein is expressed in a wide variety of normal epithelial tissues. Its distribution frequently coincides with the location of post-replicative cells within tissues. Staining patterns demonstrate that the APC protein, although often diffusely cytoplasmic in distribution, may also accumulate in the apical and immediately subapical regions, or along the lateral margins of certain cells. These results indicate that APC is significant in many tissues in addition to the colorectal epithelium. They are compatible with a function related to signalling at the adherens junction and possibly with other more complex roles in cells committed to terminal differentiation.

The p53 response to ionising radiation in adult and developing murine tissues.

The induction of the p53 response to ionising radiation has been studied during murine development and in the adult animal. The response has been assessed by precise quantitative assay of p53 protein levels in tissues and by immunohistochemistry. Newly developed transgenic mice in which a lacZ transgene is driven by a p53 response element have also been used to directly assess the transcriptional activity of the induced protein. There is striking developmental control of the p53 response so that in early development all tissues accumulate high levels of p53 following radiation and indeed p53 is present at elevated levels in some unirradiated tissues. Later in development clear heterogeneity of the p53 response becomes apparent, both in terms of the responses of individual tissues and of cell populations, within those tissues. The study of lacZ transgene expression and the occurrence of apoptosis in different tissues that accumulate p53 protein point to a further level of control regulating the nature and degree of the downstream response to elevated levels of p53 in cells. These findings have important implications for the susceptibility of different tissue types to carcinogenic and other insults. The early expression of the p53 response is consistent with novel models of p53 function that suggest it may have evolved principally as a defense against teratogenic insult that permits plasticity of development.

Modification of two distinct COOH-terminal domains is required for murine p53 activation by bacterial Hsp70.

Activation of the latent DNA binding function of human p53 protein by the bacterial Hsp70, DnaK, represents a unique reaction in which a heat shock protein can interact with a native protein to affect its function. We have localized a likely DnaK interaction site on native human p53 tetramers to a motif flanking the COOH-terminal casein kinase II and protein kinase C phosphorylation sites. Murine p53 is less efficiently activated by DnaK, which has permitted a search for factors that might cooperate in p53 activation by DnaK. We show that optimal activation by DnaK may be dependent upon the phosphorylation state of murine p53, in particular, modification of p53 at the cdc2 phosphorylation site by point mutation decreases the extent of activation by DnaK. Additionally, the monoclonal antibody PAb241, binding in the vicinity of the cdc2 phosphorylation site, is able to activate the specific DNA binding function of p53. This has led us to propose a second regulatory motif flanking the tetramerization domain of p53 that cooperates with factors binding at the negative regulatory domain in the extreme COOH terminus.

Schools, families, and early adolescents: what are we doing wrong and what can we do instead?

Although most individuals pass through adolescence without excessively high levels of "storm and stress," many individuals experience difficulty during this period. Why? Is there something unique about this developmental period that puts individuals at greater risk for difficulty? This paper focuses on these questions and advances the hypothesis that some of the "negative" psychological and behavioral changes associated with adolescent development result from a mismatch between the needs of developing adolescents and their experiences at school and at home. It provides theoretical and empirical examples of how this mismatch develops, how it is linked to negative age-related changes in early adolescents' motivation, self-perceptions, self-evaluations, and psychological competence, and how we could provide more developmentally appropriate social environments, particularly at school.