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Biomedical subjects

A Sparks

Publications and source records attributed to A Sparks.

14 recordsLinked to original sources
PubMed2001

Public health response to an incident of secondary chemical contamination at a beach in the United Kingdom.

OBJECTIVES: To gather enough data from a large scale investigation involving two health authorities, to assess the possible concentrations and routes of exposure and the consequent health implications. To use the data to decide whether a polluted beach should remain open to the public. In Spring 1997, a chemical incident came to light at a beach on the south coast of England when a local resident reported a sulphurous smell, visible signs of oil, and reduced numbers of fishing bait. The beach was situated adjacent to a former gasworks site and was accessible to the public. The incident was reported to the local authority and was initially investigated by the Environment Agency and the local authority. An Environment Agency report confirmed contamination of the beach with cyanide, ammonia, and polycyclic aromatic hydrocarbons (PAHs) with associated potential health risks. The incident was then referred to the local health authorities for investigation. METHODS: The investigation was carried out in four stages: comprehensive sampling and analysis to identify the extent of contamination, followed by an assessment of risk to health; establishment of a long term monitoring programme to identify any changes in contaminant concentrations; investigation of the effects of the contamination on shellfish; and review of the routine monitoring data and current sampling strategy. RESULTS: The initial investigation confirmed that the beach was contaminated, with the most likely source being the adjacent former gasworks site. The level of contamination was not found to be likely to pose a hazard to users of the beach. However, subsequent investigation of shellfish in the area led to warning signs being erected on the beach to prevent human consumption of mussels contaminated with PAHs. CONCLUSIONS: Several lessons can be learnt from this investigation, which can be applied to incident management more generally: the importance of collaboration and coordination; the need for early involvement of the health authority; and the importance of carrying out appropriate sampling and analysis as soon as possible, to assess the risk to health and the environment.

Ammonia↗
PubMed2000

Analysis of adenomatous polyposis coli promoter hypermethylation in human cancer.

Germ-line mutations in the tumor suppressor gene APC are associated with hereditary familial adenomatous polyposis (FAP), and somatic mutations are common in sporadic colorectal tumors. We now report that methylation in the promoter region of this gene constitutes an alternative mechanism for gene inactivation in colon and other tumors of the gastrointestinal tract. The APC promoter is hypermethylated in 18% of primary sporadic colorectal carcinomas (n = 108) and adenoma (n = 48), and neoplasia with APC methylation fails to express the APC transcript. Methylation affects only wild-type APC in 95% of cases and is not observed in tumors from FAP patients who have germ-line APC mutations. As with APC mutation, aberrant APC methylation occurs early in colorectal carcinogenesis. When other tumor types are analyzed (n = 208), methylation of the APC promoter is not restricted to the colon but is present in tumors originating elsewhere in the gastrointestinal tract but rarely in other tumors. Our data suggest that hypermethylation of APC provides an important mechanism for impairing APC function and further underscores the importance of the APC pathway in gastrointestinal tumorigenesis.

Alleles↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Defective nuclear localization of p53 protein in a Chinese hamster cell line is associated with the formation of stable cytoplasmic protein multimers in cells with gene amplification.

Many p53 functions require p53 transport into the nucleus. Mutant p53 also generally accumulates in the nucleus of transformed or neoplastic cells. However, examples of cytoplasmic accumulation of wild-type or mutant p53 have also been reported. Various explanations have been provided for defective nuclear localization. Here we propose a novel example of cytoplasmic p53 localization which occurs in cells showing gene amplification and appears to be due to the formation of stable p53 multimers. We studied a methotrexate-resistant Chinese hamster cell line (MTX M) carrying amplified dihydrofolate reductase genes and derived from a cell line with p53 nuclear accumulation. MTX M showed cytoplasmic p53 localization and, on immunoblots, several extra bands in the high molecular weight region, besides the expected 53 kDa band. p53 localization and the appearance of high molecular weight bands appeared to be correlated with the degree of DNA amplification. However, amplification of dihydrofolate reductase itself was not involved. Changing the p53 phosphorylation status quantitatively influenced the formation of high molecular weight bands. Cell fusion experiments demonstrated that p53 cytoplasmic localization in MTX M is a dominant phenotype. This result suggests that the defect causing lack of nuclear localization in this cell line does not reside in the nucleus. In the cytoplasm of MTX M and of wild-type/MTX M heterodikaryons p53 gives rise to protein complexes that are unable to re-enter the nucleus. The formation of such protein complexes is dependent on the amplification of an unknown gene product.

3T3 Cells↗
PubMed1999

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.

Biological Transport↗
PubMed1999

Transcription factor E2F-1 is upregulated in response to DNA damage in a manner analogous to that of p53.

The transcription factor E2F-1 directs the expression of genes that induce or regulate cell division, and a role for E2F-1 in driving cells into apoptosis is the subject of intense discussion. Recently it has been shown that E2F-1 binds and coprecipitates with the mouse double-minute chromosome 2 protein (Mdm2). A domain of E2F-1 (amino acids 390 to 406) shows striking similarity to the Mdm2 binding domain of the tumor suppressor protein p53. It is known that interaction of Mdm2 with p53 through this domain is required for Mdm2-dependent degradation of p53. We show here that E2F-1 protein is upregulated in response to DNA damage. The kinetics of induction are dependent upon the source of DNA damage, i.e., fast and transient after irradiation with X rays and delayed and stable after irradiation with UVC, and thus match the kinetics of p53 induction in response to DNA damage. We show further that E2F-1 is also upregulated by treatment with the transcription inhibitor actinomycin D and with the kinase inhibitor DRB, as well as by high concentrations of the kinase inhibitor H7, all conditions which also upregulate p53. In our experiments we were not able to see an increase in E2F-1 RNA production but did find an increase in protein stability in UVC-irradiated cells. Upregulation of E2F-1 in response to DNA damage seems to require the presence of wild-type p53, since we did not observe an increase in the level of E2F-1 protein in several cell lines which possess mutated p53. Previous experiments showed that p53 is upregulated after microinjection of an antibody which binds to a domain of Mdm2 that is required for the interaction of Mdm2 with p53. Microinjection of the same antibody also increases the expression of E2F-1 protein, while microinjection of a control antibody does not. Furthermore, microinjection of Mdm2 antisense oligonucleotides upregulates E2F-1 protein, while microinjection of an unrelated oligonucleotide does not. These data suggest that E2F-1 is upregulated in a similar way to p53 in response to DNA damage and that Mdm2 appears to play a major role in this pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo.

BACKGROUND: The transcriptional activation function of the p53 tumour suppressor protein is induced by DNA damage and results in growth arrest and/or apoptotic responses. A key component of this response is the dramatic rise in p53 protein concentration resulting from an increase in the protein's stability. Very recently, it has been suggested that interaction with the Mdm2 protein may target p53 for rapid degradation. We have designed a gene encoding a small protein that binds tightly to the p53-binding pocket on the Mdm2 protein. We have constructed the gene by cloning a phage display optimised Mdm2-binding peptide into the active-site loop of thioredoxin. RESULTS: When introduced into cells containing low levels of wild-type p53, this protein causes a striking accumulation of the endogenous p53 protein, activation of a p53-responsive reporter gene, and cell cycle arrest mimicking the effects seen in these cells after exposure to UV or ionising radiation. Microinjection of a monoclonal antibody to the p53-binding site on Mdm2 achieves a similar effect, establishing its specificity. CONCLUSIONS: These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein-protein interactions in cells.

Amino Acid Sequence↗
PubMed1996

Epitope analysis of the murine p53 tumour suppressor protein.

The identification and characterisation of the p53 tumour suppressor has relied extensively on the use of immunological reagents. To facilitate further characterisation of the murine p53 protein (Mp53), and its interaction with other proteins, we have characterised the antigenic sites of Mp53 in fine detail. Using an overlapping Mp53 peptide library we report the identification by Pepscan ELISA of the epitopes of nine antibodies. We have also used this technique to determine whether corresponding epitopes were present in a human p53 (Hp53) peptide library. This comparison was extended to include polyclonal sera of mice immunized with either Mp53 or Hp53, to compare the overall range of antigenic sites. The range of antigenic sites identified by polyclonal sera is very similar, although the N-terminus of Mp53 is clearly not an immunodominant region, in contrast to the N-terminus of Hp53. However, the N-terminus of Mp53 is immunogenic in rabbits as demonstrated by the Pepscan ELISA of CM5 serum (a rabbit anti-Mp53 serum used in analysing p53 expression in mice). Since, very few new antigenic sites were identified in either Mp53 or Hp53, new approaches will have to be employed to identify novel immunological reagents against human and murine p53.

Animals↗
PubMed1995

Small peptides activate the latent sequence-specific DNA binding function of p53.

Normal cells contain p53 protein in a latent state that can be activated for sequence-specific transcription by low levels of UV radiation without an increase in protein levels. Microinjection of cells with an antibody specific to the C-terminal negative regulatory domain can activate the function of p53 as a specific transcription factor in the absence of irradiation damage, suggesting that posttranslational modification of a negative regulatory domain in vivo is a rate-limiting step for p53 activation. Small peptides derived from the negative regulatory domain of p53 have been used as biochemical tools to distinguish between allosteric and steric mechanisms of negative regulation of p53 tetramer activity. Presented is the development of a highly specific peptide activation system that is consistent with an allosteric mechanism of negative regulation and that forms a precedent for the synthesis of novel low molecular mass modifiers of the p53 response.

Allosteric Regulation↗
PubMed1994

Immunochemical analysis of the interaction of p53 with MDM2;--fine mapping of the MDM2 binding site on p53 using synthetic peptides.

The function of p53 is modulated by binding to a number of cellular and viral proteins, such as MDM2 and SV40 large T antigen. An initial immunochemical characterization of the p53-MDM2 complex in a rat fibroblast cell line (Clone 6) suggested that the anti-p53 monoclonal antibody Bp53-19 failed to immunoprecipitate the complex, and only recognized a fraction of the available p53 protein. Following the recent identification of the Bp53-19 epitope at the N-terminal end of p53, in the vicinity of where MDM2 protein was known to bind, we investigated the possibility that Bp53-19 might identify a region of p53 that interacts with MDM2 protein. MDM2 was found to bind with great specificity to short synthetic peptides derived from the N-terminus of p53. Several p53 synthetic peptides libraries, and an alanine substitution series at the optimal binding site, were used to establish the MDM2 binding site, in fine detail, to the sequence TFSGLW (aa 18-23) in mouse and TFSDLW in man (aa 18-23). The key residues required for MDM2 binding are almost identical to those required for the monoclonal antibody Bp53-19 to bind and this region of p53 is recognised by many other anti-p53 antibodies.

Amino Acid Sequence↗
PubMed1988

Rape crisis program helps restore dignity and control.

A rape victim often finds herself in a hospital emergency room before she even has a chance to think about what happened, how she feels, or what she needs. Emergency rooms are not equipped to help restore the victim's feelings of safety, dignity, and control over her life. To remedy that situation, St. Vincent's Hospital and Medical Center of New York instituted the Rape Crisis Program in 1977. The program, staffed by two professional social workers, an administrative assistant, and a student doing field work, offers counseling as an enabling, healing process in which the client actively participates. Two aspects of the counseling are particularly important: providing a perspective on the recovery process and helping the survivor discuss details of the assault that she would not want to tell anyone else. Volunteers assist victims in the emergency room at night and on weekends after completing eight training sessions of 2 1/2 hours each. Readings, discussion, and role playing help trainees understand the fear, anger, and shame that sexual assault victims experience. Sometimes victims return to help others by becoming volunteers.

Counseling↗
PubMedDate not supplied

Unusual luminescent properties of odd- and even-substituted naphthyl-derivatized dioxetanes.

With the advent of enzymatically induced chemiluminescence and improved instrumentation for luminometry, ultrasensitive detection of a wide variety of analytes is now possible using standard immunoassay and DNA probe formats. Model molecular orbital calculations and literature precedent suggest that the singlet efficiencies observed upon decomposition of dioxetanes appended with donor substituted aromatic moieties are dependent on substitution pattern. We have recently discovered, in a series of 3-(2'-spiroadamantane)-4-methoxy-4-acetoxynaphth-2'-yl-1,2-+ ++dioxetanes, that enzymatic generation of a non-conjugated, charge transfer excited state results in luminescence of markedly different properties than that observed from an isomeric, conjugated excited state. An example of the former type, 3-(2'-spiroadamantane)-4-methoxy-4-(7"-acetoxy)naphth-2'-yl- 1,2- dioxetane (1) emitting at 550 nm, not only provides an increase in phi CL, but exhibits a dramatic bathochromic shift of 80-110 nm from the 460 nm emission of the conjugated isomer 3-(2'-spiroadamantane)-4-methoxy-4-(6"-acetoxy)naphth-2'-yl- 1,2-dioxetane (2). These developments, along with the attendant glow-type luminescence kinetics displayed during the enzymatic decomposition of the new 'odd-pattern' dioxetane, allow the design of simple protocols capable of simultaneous or 'multichannel' detection of several analytes.

DNA Probes↗