The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Serial propagation of human ovarian surface epithelium in tissue culture

Most human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 micrograms/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20-25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin-positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin-positive form in medium with EGF/HC. OSE cells grown in WM/5-15% FBS were epithelial and near-stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.     

An agent or agents produced by virus-transformed cells cause unregulated ruffling in untransformed cells

KNRK cells (a normal rat kidney [NRK] cell line transformed by Kirsten murine sarcoma virus) in sparse culture exhibit a highly ruffled morphology, but the cause of this ruffling is unknown. In this study, we have demonstrated that the continuous, excess ruffling on KNRK cells is caused by one or more soluble agents secreted by the KNRK cells themselves. To do this study, an assay for ruffling responses in live cell cultures was defined, and its reproducibility was demonstrated. This assay permitted observation of the kinetics of ruffling responses (percentage of cells ruffled as a function of time after stimulation). This method was used to compare the kinetics of ruffling induced by insulin, epidermal growth factor, fibroblast growth factor, glucose, and KNRK cell conditioned medium (CM). Ruffling was elicited on NRK cells by each of the polypeptide mitogens and nutrients, but, in each case, this ruffling subsided spontaneously within an hour. CM from KNRK cells also caused ruffling movements on untransformed NRK cells, but this ruffling continued for at least 20 h. This response was largely blocked by premixing the KNRK cell CM with rabbit IgG against rat transforming growth factor, type alpha, (TGF-alpha). KNRK cells made quiescent (ruffle free) by a pH shift (from 7.4 to 8.4) responded to insulin, glucose, and KNRK cell CM with kinetics similar to those observed for each of these factors in NRK cells. The unusual feature for the ruffle-inducing agent(s) produced by KNRK cells was that this activity was not subject, in either NRK or KNRK cells, to the cellular off-regulation that limits the responses to insulin or glucose. Thus, the continuous ruffling of KNRK cells is caused by their own unregulated ruffle-inducing agent or agents, which appear to include TGF-alpha. This work also demonstrates that kinetic analysis of cellular responses to exogenous factors can provide new insights into the regulatory mechanisms involved in the normal limitation of these responses.     

Identification of Glycolytic Enzyme Polypeptides on the Two-Dimensional Protein Map of Saccharomyces cerevisiae and Application to the Study of Some Wine Yeasts

Using a modification of the basic two-dimensional polyacrylamide gel electrophoresis technique, the polypeptides of the protein map of Saccharomyces cerevisiae involved in glycolysis were investigated. This study resulted in a reassignment of two of the seven glycolytic enzyme polypeptides previously identified (Ludwig et al., Mol. Cell. Biol. 2:117-126, 1982), those corresponding to phosphoglycerate kinase and to alcohol dehydrogenase. It also resulted in the identification of two additional glycolytic polypeptides, the enolase B monomer and the glyceraldehyde phosphate dehydrogenase B monomer. The glycolytic enzymes polypeptides so identified were investigated in 5 laboratory strains (all S. cerevisiae) and in 11 commerical strains used for wine making (S. cerevisiae and Saccharomyces bayanus). It appeared highly significant that a particular electrophoretic variant of the glyceraldehyde phosphate dehydrogenase B monomer was found only in the wine yeasts. Furthermore, it was strongly suggested that S. cerevisiae and S. bayanus strains are distinguishible on the basis of a different electrophoretic migration of the enolase B monomer.     

Coexistence of incompatible plasmids in a bacterial population living under a feast and famine regime

A model is formulated to examine the possibility of (co)existence of plasmids of the same incompatibility and surface exclusion group in a bacterial population living under a feast-and-famine regime. The condition is given under which a growth rate decreasing plasmid can invade a bacterial population. It appears that in case only one plasmid type is present, the frequency of plasmid bearers will tend to a stable equilibrium if the food supply at each growth site gets exhausted and if both plasmid-free and plasmid-bearing bacteria need an equal quantity of food per cell division. If these two conditions are not satisfied, the frequency of plasmid-bearers might oscillate. Two plasmids will sometimes be able to coexist, but only if they follow different survival strategies; one with a high conjugational transfer rate and a lower fitness of its host, and the other with a low transfer rate and a higher host fitness. Coexistence of three plasmids of the same surface exclusion group is impossible.     

Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus

Summary The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX₂CysX₄HisX₄Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX₂CysX₄HisX₄Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz ¹H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747±0.209 Å for backbone atoms and 1.709±0.187 Å when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr²⁸, Trp³⁵, Lys³⁷, Lys⁴¹ and Lys⁴² residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.     

Effect of heat stress on glucose kinetics during exercise

Hargreaves, Mark, Damien Angus, Kirsten Howlett, Nelly MarmyConus, and Mark Febbraio. Effect of heat stress on glucose kinetics during exercise. J. Appl.Physiol. 81(4): 1594-1597, 1996.To identify themechanism underlying the exaggerated hyperglycemia during exercise inthe heat, six trained men were studied during 40 min of cyclingexercise at a workload requiring 65% peak pulmonary oxygen uptake(O_{2 peak}) on twooccasions at least 1 wk apart. On one occasion, the ambient temperaturewas 20°C [control (Con)], whereas on the other, it was40°C [high temperature (HT)]. Rates ofglucose appearance and disappearance were measured by using a primedcontinuous infusion of[6,6-²H]glucose. Nodifferences in oxygen uptake during exercise were observed betweentrials. After 40 min of exercise, heart rate, rectal temperature,respiratory exchange ratio, and plasma lactate were all higher in HTcompared with Con (P < 0.05). Plasmaglucose levels were similar at rest (Con, 4.54 ± 0.19 mmol/l; HT,4.81 ± 0.19 mmol/l) but increased to a greater extent duringexercise in HT (6.96 ± 0.16) compared with Con (5.45 ± 0.18;P < 0.05). This was the result of ahigher glucose rate of appearance in HT during the last 30 min ofexercise. In contrast, the glucose rate of disappearance and metabolicclearance rate were not different at any time point during exercise.Plasma catecholamines were higher after 10 and 40 min of exercise in HTcompared with Con (P < 0.05),whereas plasma glucagon, cortisol, and growth hormone were higher in HTafter 40 min. These results indicate that the hyperglycemia observedduring exercise in the heat is caused by an increase in liver glucoseoutput without any change in whole body glucoseutilization.