Epitope analysis of the human p53 tumour suppressor protein

Allogenic, preserved cartilage is often used for reconstruction of face. This study was undertaken to analyze the effects of cartilage transplantation. In analyzed group of 437 patients after cartilage transplantation, 42.2% were operated because of posttraumatical changes, 29.0% because of congenital malformations. In 16.7% nonspecific inflammations were the cause of reconstructive operations. Malformations were mainly localised in nose 59%, ear concha 16.5% and mandible 10.9%. Human costal cartilage, preserved in 0.9% NaCl and radiation-sterilized was used for reconstruction. 24-190 months after surgery (in several clinical units) patients were examined and results were collected in special questionnaire by the team who performed surgery. The results of treatment were compared with age, diagnosis and localisation of changes. It was found that very good result of treatment was achieved in 33.5% of patients, in 41.8% result was satisfactory. In 19.9% of operated patients result of treatment was unsatisfactory. Correlation of some clinical and biological characteristics with the result of treatment is discussed.     

Baculovirus p33 binds human p53 and enhances p53-mediated apoptosis

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.     

In vitro pattern formation during neurogenesis in neuroectodermal progenitor cells immortalized by p53-deficiency

In vitro neural differentiation was induced in a p53-deficient immortalized neuroectodermal progenitor cell line, NE-4C, by treatment with retinoic acid [K. Schlett and E. Madarász (1997) J. Neurosci. Res. 47, 405-416]. Rearrangement of nestin filaments was an early marker of neuron-formation. The increase in neurofilament protein content was accompanied by a decrease in the expression of nestin filaments in induced precursors. Cells with astroglial features appeared with a delay of 4-5 days compared to the appearence of neurons. Future neurons were sorted out from the substrate-attached population of apparently non-induced cells. The sorting out of future neurons resembled the separation of neural precursors in vivo. The continuous changes in the shape and also in the position of the cells resulted in the formation of characteristic morphological patterns. On the basis of morphological changes, five characteristic stages of in vitro neural differentiation were distinguished. The analysis of the morphological changes revealed that cell-to-cell interactions played an essential role in the cell fate decision made by induced precursors. Our observations indicate that the NE-4C cell line can serve as an in vitro model to investigate some early steps of neurogenesis.     

The time-related expression of p53 protein in human skin wounds--a quantitative immunohistochemical analysis

Failure to acknowledge ways of knowing in nursing education curricula other than linear reasoning hinders the development of the full extent of mental abilities brought to learning situations by nursing students. Nurse educators are challenged to develop creative methods to facilitate nursing students' intuitive thinking. In this article, a teaching strategy is described in which graduate students' exemplars of intuition in clinical practice are shared with undergraduate nursing students. Implications of using this teaching approach to demystify the intuitive process and address its legitimacy are discussed.     

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G1 and G2/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.     

In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA

The 790 loop is a conserved hairpin located between positions 786 and 796 of Escherichia coli 16 S rRNA that is required for ribosome function. Using a novel genetic approach, all positions in the loop were simultaneously mutated and functional mutant sequences were selected in vivo. This "instant evolution" experiment revealed that approximately 190 of the 262,144 possible mutant sequences were functional. Analysis of functional mutant sequences allowed discrimination between nucleotides directly involved in protein synthesis and those involved primarily in loop structure. Among the functional mutant sequences, positions 789 and 791 were invariant and extensive covariation was observed among the nucleotides at the base of the loop at positions 787, 788, 794 and 795. NMR and thermodynamic analyses of model 790 hairpins in vitro revealed weak pairing interactions between positions 787 and 795 and between positions 788 and 794 consistent with the in vivo mutational analysis. Functional analysis of site-directed mutants containing all possible nucleotide combinations at positions 787 and 795 in vivo showed that stable base-pairs at these positions prevent subunit association.     

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.     

In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Deltap6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Deltap6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Deltap6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Deltap6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Deltap6 and nucleic acid are not sufficient for the formation of normal-sized particles.     

Involvement of p53 gene mutations in human endometrial carcinomas

Mutations in the p53 gene are associated with a wide variety of human malignancies. Point mutation in one allele and loss of the remaining one generally lead to inactivation of p53 protein. A high frequency of allelic losses corresponding to the 17p13.3 region that contained the p53 gene sequence was also noted in human endometrial carcinoma. Thus, in order to confirm involvement of the p53 gene in endometrial carcinogenesis, we searched for nucleotide sequence change in this gene in 42 endometrial carcinomas that had been subjected to previous LOH analyses. Using the polymerase-chain-reaction-single-strand conformation polymorphism (PCR-SSCP) method, we detected p53 gene mutations in 4 specimens. Two adenocarcinomas with allelic loss on 17p contained a mutant p53 gene in the allele that was retained. One specimen with a p53 gene mutation contained a 17q deletion but was uninformative for LOH on 17p. p53 gene mutation was also noted in the remaining stage-I carcinoma, though the 17p deletion was not detected in the previous LOH examination. However, 5 specimens with the LOH on 17p retained the wild-type p53 gene. In the remaining 33 specimens, both alleles of p53 gene seemed to be normal. The mutations observed in 2 specimens (cases 10 and 24), involving C-to-T and T-to-G substitutions, were located in a highly conserved region. However, the mutations identified in the remaining 2 cases (29 and 35) were at regions positioned outside conserved stretches.     

Analysis of p53 in human cutaneous melanoma cell lines

Mutations in the p53 tumour suppressor gene have been detected in a variety of human malignancies. Mutations have been found predominantly in conserved regions two to five. Our aim was to analyse p53 at the protein and DNA level in seven melanoma cell lines of cutaneous origin (HMB-2, DX3, LT5.1, MJM, SK23, A375P and A375M), including two parental/metastatic derivatives (A375P and A375M; DX3 and LT5.1). By immunohistochemical staining with three mouse monoclonal antibodies and a rabbit polyclonal serum, it was possible to observe differential nuclear expression of p53. The quantitation of p53 protein levels by ELISA correlated with the nuclear staining pattern. Western blotting showed an intact p53 protein in all cell lines; p53 was polymorphic in three cell lines (MJM, A375P and A375M). DNA sequencing studies showed that all cell lines had wild type p53. These results suggest that p53 is unlikely to play a significant role in the genesis of cutaneous melanoma.     

In vitro association of the phosphatidylinositol 3-kinase regulatory subunit (p85) with the human insulin receptor

The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its beta-subunit. The 85 kDa regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85), an SH2 domain protein, has been implicated as a regulatory molecule in the insulin signal transduction pathway. For the present study, glutathione S-transferase (GST) fusion proteins of p85 SH2 domains were used to determine if such motifs associate directly with the autophosphorylated human insulin receptor. The p85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associate with the autophosphorylated beta-subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C-gamma 1 (PLC gamma 1) N + C SH2 domains exhibited measurable affinity for the activated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed efficiently by a 15-residue tyrosine-phosphorylated peptide corresponding to the carboxyl-terminal region of the insulin receptor, but not by phosphopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N + C SH2 domain-insulin receptor complex with an IC0.5 of 2.3 +/- 0.35 microM, whereas a 10-residue phosphopeptide derived from the insulin receptor substrate 1 (IRS-1) competed with an IC0.5 of 0.54 +/- 0.10 microM. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl-terminal region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/insulin receptor complex may lead to signaling pathways independent of IRS-1.     

Adenovirus-mediated wild-type p53 gene transfer and overexpression induces apoptosis of human glioma cells independent of endogenous p53 status

A role for Zn2+ in a variety of neurological conditions such as stroke, epilepsy and Alzheimer's disease has been postulated. In many instances, susceptible neurons are located in regions rich in Zn2+ where nerve growth factor (NGF) levels rise as a result of insult. Although the interaction of Zn2+ with this neurotrophin has previously been suggested, the direct actions of the ion on NGF function have not been explored. Molecular modeling studies predict that Zn2+ binding to NGF will induce structural changes within domains of this neurotrophin that participate in the recognition of TrkA and p75NTR. We demonstrate here that Zn2+ alters the conformation of NGF, rendering it unable to bind to p75NTR or TrkA receptors or to activate signal transduction pathways and biological outcomes normally induced by this protein. Similar actions of Zn2+ are also observed with other members of the NGF family, suggesting a modulatory role for this metal ion in neurotrophin function.