Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells.

Several mutant, but not wild-type, p53 proteins form complexes with hsp72/73 heat shock-related proteins in simian virus 40-transformed monkey COS cells. We carried out a detailed biochemical and structural mapping analysis of p53 and report here that p53-hsp72/73 complex formation showed considerable structural specificity. Such complexes were remarkably stable, but unlike analogous complexes formed between p53 and simian virus 40 T antigen, they did not form in in vitro association assays. p53-hsp72/73 complex formation in vivo appears to be dependent on aspects of mutant p53 protein conformation. However, absence of the conformation-sensitive epitope recognized by monoclonal antibody PAb 246 was not reliably diagnostic of such complexes, nor was p53-hsp72173 binding reliably diagnostic of oncogenic activation.

Defining the minimal requirements for papilloma viral E6-mediated inhibition of human p53 activity in fission yeast.

The majority of human anogenital carcinomas show evidence of papillomavirus infection. To facilitate viral replication, viruses disable key cellular responses which would otherwise precipitate cell suicide. An obligate factor in one such response is the p53 tumour suppressor protein. p53 gene mutation is an infrequent event in anogenital cancer, apparently due to the action of HPV E6 protein, which inhibits wild-type p53 function by stimulating the degradation of p53 protein. p53 is required for the apoptotic response that is triggered in untransformed cells following inappropriate cell-cycling. E6 directed inhibition of p53 function thus facilitates the survival of transformed cells. We have developed a genetically tractable model that reports E6 protein-mediated human p53 inactivation in the fission yeast Schizosaccharomyces pombe. Functional dissection of the requirements for E6 directed inhibition in this system reveal an absolute requirement for the presence of both E6 protein and the human E3 ubiquitin ligase, E6-AP. Using a defined set of E6 mutants we show that degradation of p53 protein rather than E6/p53 association is likely required for E6-mediated inhibition. This S. pombe based system represents a candidate screen for novel antiviral agents that act by disrupting the E6/E6-AP/p53 interaction.

Human p53 directs DNA strand reassociation and is photolabelled by 8-azido ATP.

p53 is the most frequent known target for mutation in human cancer. Evidence suggests that p53 protein may be involved variously in transcription and cell cycle control, in DNA replication and in G1 checkpoint control following the cellular response to radiation induced DNA damage. p53 blocks DNA replication of the small DNA tumour virus, simian virus 40, by inhibiting unwinding of the viral origin of replication by the DNA helicase activity of the virally encoded large T antigen protein. Here we report the novel observation that human p53 protein can bind ATP and exhibits an intrinsic ATP stimulated DNA strand reassociation activity. Both activities map to the carboxyl terminal 128 amino acids of p53. Thus, in addition to any role in transcription, our results indicate that p53 is potentially capable of inhibiting mammalian replicative DNA synthesis by blocking the DNA strand separation step during replication origin recruitment. However, the ability of p53 to modulate the topological relationship between complementary nucleotide strands is also compatible with a direct role for p53 in other aspects of DNA synthesis, recombination or repair.

Human p53 binds DNA as a protein homodimer but monomeric variants retain full transcription transactivation activity.

Wild-type human p53 protein is able to self-associate and consists predominantly of homotetramers in solution. In earlier work we identified the protein sequence motifs involved in p53 quaternary structure and showed that while monomeric p53 protein retains tumour suppressor function, monomeric tumour mutant p53 lacks dominant transforming activity. In this report we use point mutated and truncated cDNA genes encoding self-association defective human p53 proteins to investigate the relationship between p53 protein quaternary structure and the associated activities of transcription transactivation and target specific DNA binding. We show that p53 binds to a target oligonucleotide as a protein homodimer and that p53 dimerisation is required for detectable DNA binding. We found no evidence for p53 tetramer: DNA complexes and we suggest that the quaternary structure status of p53 may regulate a DNA binding associated activity. Monomeric p53 proteins failed to bind DNA in these assays but exhibited increased transactivating activity. Thus, both transcription transactivation and tumour suppressor functions act independently of p53 protein self-association and DNA binding. We propose that our results validate the p53 dimerisation motif as a target for rational anticancer drug design. We predict that compounds able to block p53 dimer assembly would inhibit the dominant transforming activities of mutant p53 in tumours retaining expression of a mutant allele, while leaving intact the wild-type p53 associated activities of transcription transactivation and transformation suppression in unaffected tissue.

The p53 nuclear localisation signal is structurally linked to a p34cdc2 kinase motif.

We have identified a region of human p53 protein with striking homology to a sequence motif on Simian Virus 40 T antigen which includes the nuclear localisation signal. Mutation of basic amino acid residues in this region of p53 (residues 312 to 323; SSSPQPKKKP) compromises transport of p53 protein to the nucleus. The sequence functions efficiently as a nuclear localisation signal when fused to E. coli beta galactosidase. Serine 315 within this p53 structural motif is phosphorylated in vitro by the cell cycle kinase p34cdc2. Thus in both T antigen and p53, nuclear localisation signal and p34cdc2 kinase acceptor residue map to a contiguous region of primary amino acid sequence.

Endogenous HeLa p53 proteins are easily detected in HeLa cells transfected with mouse deletion mutant p53 gene.

It has been reported [Matlashewski et al. (1986). Eur. J. Biochem., 154, 665-672] that HeLa cells contain no detectable p53 protein, although they contain p53 mRNA which is translationally active. Here it is shown that endogenous HeLa p53 proteins were easily detected in HeLa cells transiently expressing mouse deletion mutant p53 gene after transfection with the appropriate recombinant plasmid. This detection was obtained by immunoprecipitation coupled with SDS-PAGE as well as by Western blotting experiments. Our results strongly suggest that HeLa p53 mRNA is actually translated in vivo, generating an extremely unstable p53 protein. Considering that the HeLa cell line is a HPV-18-positive human cervical carcinoma cell line, this high instability of HeLa p53 proteins is in keeping with the finding that E6 oncoprotein encoded by human papillomavirus 16 or 18 promotes the degradation of p53 proteins [Scheffner et al. (1990). Cell, 63, 1129-1136].

A "no-hybrids" screen for functional antagonizers of human p53 transactivator function: dominant negativity in fission yeast.

We have developed a functional "no-hybrids" screen in the fission yeast Schizosaccharomyces pombe based on the transcription transactivator activity of human p53. The screen can be used to identify antagonizers and modulators of p53 activity. Expression of functional full-length human p53 is conditionally lethal to the screen reporter strains. Co-expression of specific inhibitory proteins promotes cell survival and growth. We have validated the "no-hybrids" system by (a) successful modeling of human wild-type p53 interaction with SV40 large T antigen, Mdm2 and a panel of tumor-derived human p53 mutants, (b) demonstrating the screening system's efficiency through identification of a dominant negative fragment of p53 itself in a library screen context and (c) using Drosophila p53 to demonstrate that the system can detect evolutionarily distant p53 homologues based on their transactivator activity. The "no-hybrids" screen will be of utility in searches for p53 function-modulators of both cellular and viral origin.

Oligomerisation of full length p53 contributes to the interaction with mdm2 but not HPV E6.

The tumour suppressor protein p53 normally functions as a tetramer in a defined conformational state. Mutations within p53 which contribute to cancer development frequently induce a conformational shift in the protein which correlates with loss of wild type growth suppressor functions. Both the cell encoded mdm2 protein and the human papillomavirus oncoprotein E6 can regulate p53 function and we have examined the interaction of these proteins with p53. The E6/p53 association is sensitive to conformational alterations in the p53 protein, although oligomerisation is not necessary for this interaction to occur. Analysis of C-terminal p53 truncations has indicated that the region between residues 327 and 347 may play a role in E6 binding. Since monomeric forms of p53 retain transcriptional and transformation suppressor activities, our results indicate that E6 targets p53 proteins which retain these wild type functions. Conversely, the interaction of p53 with mdm2 is not dependent on the conformation of the p53 protein but is significantly impaired by loss of quaternary structure. It is possible that mdm2 plays a role in mediating activities of p53 which, unlike transcriptional activation, depend on oligomerisation.

Mutant p53 proteins bind hsp 72/73 cellular heat shock-related proteins in SV40-transformed monkey cells.

We have examined the expression of a series of mouse mutant, as well as wild-type, p53 proteins in SV40-transformed monkey COS cells. Wild-type mouse p53 binds predominantly to SV40 large T antigen in these cells. However, several of the mutants co-precipitate exclusively with proteins of approximately 68 Kd relative molecular mass. We show by immunological and proteolytic mapping techniques that these proteins are identical to the hsp 72/73 heat shock proteins. p53 mutants in complex with hsp 72/73 have an altered subcellular location compared to the wild-type protein and the hsp 72/73 binding p53 mutants fail to exhibit an epitope recognized by monoclonal antibody PAb 246. The existence of at least two antigenically distinct subclasses of hsp 72/73 complexed to mutant p53 is shown.

Mouse p53 blocks SV40 DNA replication in vitro and downregulates T antigen DNA helicase activity.

Immunopurified mouse p53 proteins were used to gain experimental access to the mechanisms underlying nonprimate p53 directed suppression of SV40 origin directed DNA replication in vivo. In replication competent HeLa cell extracts containing exogenous T antigen, mouse p53 blocks T antigen dependent DNA synthesis as in vivo. However, in transcription competent HeLa extracts, mouse p53 has no effect either on overall transcription or on the ability of immunopurified T antigen to downregulate SV40 early transcription. We show that although mouse p53 has no significant effect on T antigen encoded activities such as ATPase and DNA binding, helicase activity is somewhat reduced suggesting that the in vivo suppression by mouse p53 of SV40 replication may be due, at least in part, to direct modulation of T antigen function.

Selective loss of endogenous p21waf1/cip1 induction underlies the G1 checkpoint defect of monomeric p53 proteins.

Wild-type p53 protein displays a spectrum of activities including the ability to suppress transformed cell growth to direct apoptotic cell death and to mediate G1 checkpoint in response to cellular DNA damage. Earlier work showed that a self-association defective p53 protein retained transformation suppressor activity in rat embryo fibroblast based assays, but that monomerisation of tumour mutant p53 proteins resulted in loss of dominant transforming activity. In order to acquire a more detailed understanding of the biological consequences attendant on disruption of p53:p53 association we have carried out a study of the wild-type-like activities that are retained by monomeric p53 proteins and which are associated with the suppression of transformation. Here we show that monomeric p53 proteins are G1 checkpoint defective. Although able to stimulate transcription via a p53 DNA binding motif from the p21waf1/cip1 gene promoter in episome based assays these p53 proteins are unable to transactive the chromosomal p21waf1/cip1 gene and are sensitive both to degeneracy of consensus binding site and to half site spacing. Monomeric p53 proteins fail to trigger apoptosis in a BRK cell line transformed with E7 and ras. However, they retain wild type transformation suppressor activity in BRK cell based transformation assays. Our results indicate that p21waf1/cip1 induction and all related p53 dependent G1 checkpoint activities are dispensable for the p53 directed suppression of transformed cell growth, and that such transformation suppression by monomeric p53 proteins may occur in the absence of an apoptotic response.

Transcriptional activation plays a role in the induction of apoptosis by transiently transfected wild-type p53.

The p53 tumor suppressor gene has been implicated in the induction of apoptosis in several cell systems. We have recently reported than transiently-transfected wt p53 is capable of inducing apoptosis in certain transformed cell lines. We demonstrated by quantitative analysis using flow cytometry that apoptosis was restricted to the population expressing wt, but not mutant, p53. In the present study we use this model system to analyse the functional domain of p53 in the induction of apoptosis. Several constructs expressing mutations or deletions in the C-terminal oligomerization domain, the N-terminal transactivation domain or the central DNA-binding domain were introduced into HeLa cells, and the ability of the expressed proteins to induce apoptosis was evaluated. All the functional domains were found to be necessary for the induction of apoptosis. In addition, cycloheximide and actinomycin D inhibited wt p53-induced apoptosis. We therefore conclude that p53 acts in this cell system, at least in part, as a transcriptional activator in the induction of apoptosis.

A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization.

The p53 gene product has been implicated in both human and animal tumorigenesis. p53 forms heterologous complexes with the transforming proteins encoded by several different DNA tumor viruses. p53 also assembles into stable homo-oligomers. We demonstrate that the major structural determinant for the tetramerization of p53 is an alpha-helical plus basic region motif near the C-terminus of the protein. A monomeric p53 mutant adopts a conformation distinct from both 'wild-type' and 'mutant' form as defined by PAb1620 and PAb240 monoclonal antibody recognition. Nevertheless, monomeric and dimeric mutant p53 proteins retain the ability to suppress SV40 origin-directed DNA replication in vivo. Thus, p53-p53 interaction and expression of the PAb1620 epitope is not a prerequisite for such activity. We present data suggesting that suppression of replication by p53 may occur by a mechanism that is independent of detectable p53-T antigen association.

Inhaled PAF fails to induce airway hyperresponsiveness to methacholine in normal human subjects.

The effects of three increasing doses of platelet-activating factor (PAF) on airway caliber and methacholine bronchial responsiveness were studied. On separate occasions nine normal subjects inhaled a single cumulative provocation concentration of methacholine (control) causing a 40% fall (PC40 Vp30) in maximum expiratory flow rate at 70% of base-line vital capacity below total lung capacity during a partial forced expiratory maneuver or 100 or 200 micrograms PAF, and seven subjects inhaled a further dose of 400 micrograms PAF. Methacholine responsiveness was measured before, at 3 and 7 h, then on days 1, 2, 3, 4, 7, 10, and 14 after each challenge. The maximum falls in Vp30 appeared dose dependent, but a significant difference between the magnitude of the responses was only observed between the 400- and 100-micrograms PAF dose (P less than 0.05). During the control period repeated methacholine challenges resulted in a progressive increase in cumulative provocation concentration of an agonist causing a 20% fall in forced expiratory volume in 1 s from base line, reaching significance on days 1 and 2 (2.44- and 2.4-fold of base line, respectively, P less than 0.01) before returning to base line on day 7. No difference was seen in methacholine responsiveness after any of the three doses of PAF compared with that after the control. We conclude that PAF causes dose-dependent bronchoconstriction but does not change airways responsiveness to methacholine and that repeated high-dose methacholine challenge leads to loss of responsiveness to this agonist.

Pavlovian conditioning of agonistic behavior in male threespine stickleback (Gasterosteus aculeatus).

The red coloration of male stickleback (Gasterosteus aculeatus) possesses signal value in male-male interactions. Therefore, it was predicted that males would learn to associate a red signal more readily than a green signal with a conspecific rival in a Pavlovian conditioning experiment. Males were presented red and green signal lights where one signal was always paired with presentation of a rival (excitatory conditioned stimulus, CS+) and one signal was never paired with presentation of a rival (nonreinforced stimulus, CS-). Males learned the task rapidly, showing conditioned approach and zigzag responses, but CS+ vs. CS- differentiation persisted, even after a prolonged extinction period. In addition, there were no differences in learning rates between fish trained to the red signal as the CS+ and fish trained to the green signal as the CS+. The results suggest that, although males may rapidly learn about rivals, they are not predisposed to associated red (over green) with the appearance of a rival under the conditions of this experiment. Because males must establish and maintain territories in order to nest and mate, learning about neighboring rivals may be an adaptive mechanism by which males more effectively defend their territories and thereby increase their reproductive fitness.

Cost-effective evaluation and management of the acute abdomen.

1. Managed care gate-keeper financial responsibility must be balanced with specialist diagnostic and therapeutic responsibility to maximize cost effectiveness and quality of medical service. 2. Cost in health care is closely tied to length of stay and operating room time; extremes of patient age are associated with increased costs. 3. Through years of training and subsequent experience, surgeons are best qualified to direct work-up of patients with an acute abdomen, especially when the work-up requires costly imaging (e.g., CT scan, ultrasonography, angiography). 4. Cost is increased when the surgeon is not involved early in cases of acute abdomen. 5. Surgeons must be willing to enter the evaluation phase of the patient with an acute abdomen patient early and follow during the observation period to best reduce unnecessary laboratory work or tests. 6. Health care teams willing to implement pathways and then document the effect such pathways exert will be most successful in assessing new technologies' cost effectiveness.

A primigravida allegedly allergic to local anaesthetics.

Pregnant patients may give a history of allergy to local anaesthetics, but many of these supposed allergies have not been investigated. There is cross-reactivity between the amide local anaesthetics, which are the only group available in the UK for regional analgesia. We report the management of a primigravida who gave a history of allergy to two local anaesthetics, lidocaine and prilocaine. She was admitted to the labour ward at 38 weeks' gestation for challenge testing to the amide local anaesthetic bupivacaine, with full resuscitation and delivery facilities available. The tests proved negative and she was allowed home. She was re-admitted at term in labour and requested epidural analgesia. Epidural bupivacaine with fentanyl was used without incident for labour and forceps delivery. Subsequent allergy testing to lidocaine also proved negative.

Effects of tutoring in phonological and early reading skills on students at risk for reading disabilities.

This study examined the effectiveness of nonprofessional tutors in a phonologically based reading treatment similar to those in which successful reading outcomes have been demonstrated. Participants were 23 first graders at risk for learning disability who received intensive one-to-one tutoring from noncertified tutors for 30 minutes, 4 days a week, for one school year. Tutoring included instruction in phonological skills, letter-sound correspondence, explicit decoding, rime analysis, writing, spelling, and reading phonetically controlled text. At year end, tutored students significantly outperformed untutored control students on measures of reading, spelling, and decoding. Effect sizes ranged from .42 to 1.24. Treatment effects diminished at follow-up at the end of second grade, although tutored students continued to significantly outperform untutored students in decoding and spelling. Findings suggest that phonologically based reading instruction for first graders at risk for learning disability can be delivered by nonteacher tutors. Our discussion addresses the character of reading outcomes associated with tutoring, individual differences in response to treatment, and the infrastructure required for nonprofessional tutoring programs.

Development of a school building model for educating students with handicaps and at-risk students in general education classrooms.

This study, conducted in the context of a 4-year project to redesign special and remedial services in an elementary school, examined the effects of cooperative learning, cross-age tutoring, and in-class services for students with handicaps and remedial students. All students (524) in Grades 1 through 6 in two medium-sized elementary schools took part in the study. All three treatments were introduced into one of the schools, with the second school serving as a control. The cooperative learning treatment was delivered to all sixth-grade students, cross-age tutoring to special and remedial students in Grades 1 through 3, and the in-class services to all grade levels. Results indicated that, although the character of the instructional services changed markedly, none of the three treatments had much impact on achievement. Reasons for the findings are explored.

Specialized instruction within general education: a case study of one elementary school.

This study examined how classroom teachers in one elementary school approached the problem of designing specialized instruction for students with reading problems. We asked 12 teachers to rate their confidence in designing effective interventions and then to diagnose a student's reading problem, select an intervention, and implement it. Teachers were moderately confident about their ability to design effective interventions; but some teachers expressed doubts about how to proceed. Their approach to intervention differed somewhat from that of special education resource teachers. Some teachers experienced considerable difficulty in implementing the interventions they selected.