The C-terminal regulatory domain of p53 contains a functional docking site for cyclin A

Radiation injury to cells enhances C-terminal phosphorylation of p53 at both Ser315 and Ser392 in vivo, suggesting the existence of two cooperating DNA damage-responsive pathways that play a role in stimulating p53-dependent gene expression. Our previous data has shown that cyclin A-cdk2 is the major enzyme responsible for modifying p53 at Ser315 in vivo after irradiation damage and in this report we dissect the mechanism of cyclinA-cdk2 binding to and phosphorylation of p53. Although cyclin B(1)-dependent protein kinases can phosphorylate small peptides containing the Ser315 site, cyclin A-cdk2 does not phosphorylate such small peptides suggesting that additional determinants are required for cyclin A-cdk2 interaction with p53. Peptide competition studies have localized a cyclin A interaction site to a Lys381Lys382Leu383Met384Phe385 sequence within C-terminal negative regulatory domain of human p53. An alanine mutation at any one of four key positions abrogates the efficacy of a synthetic peptide containing this motif as an inhibitor of cyclin A-cdk2 phosphorylation of p53 protein. Single amino acid mutations of full-length p53 protein at Lys382, Leu383, or Phe385 decreases cyclin A-cdk2 dependent phosphorylation at Ser315. Cyclin B(1)-cdk2 complexes are not inhibited by KKLMF motif-containing peptides nor is p53 phosphorylation by cyclin B-cdk2 reduced by mutation of the cyclin A interaction site. These data identifying a KKLMF cyclin A docking site on p53 protein highlight a common cyclin A interaction motif that is shared between the tumour suppressor proteins pRb and p53.     

Binding properties of a highly potent and selective iodinated aminopeptidase N inhibitor appropriate for radioautography

Aminopeptidase N (APN) is a zinc metallopeptidase involved in the inactivation of biologically active peptides. The knowledge of its precise distribution is crucial to investigate its physiological role. This requires the use of appropriate probes such as the recently developed highly potent and selective radiolabeled APN inhibitor 2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ (12 5)I]iodotyrosine ([(125)I]RB 129). Its binding properties were investigated using rat brain homogenates (K(d)=3.4 nM) or APN expressed in COS-7 cells (K(d)=0.9 nM). The specific binding was 95% at [K(d)], and preliminary autoradiography in intestine is promising. The decreased affinity of [(125)I]RB 129 (=10(-6) M) for the E(350)D APN mutant, supports the critical role of E(350) in the amino-exopeptidase action of APN.     

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.     

Marsupials and monotremes sort genome treasures from junk

A recent landmark paper demonstrates the unique contribution of marsupials and monotremes to comparative genome analysis, filling an evolutionary gap between the eutherian mammals (including humans) and more distant vertebrate species.     

Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human gamma delta T cells

Small phosphorylated metabolites from mycobacteria stimulate human gammadelta T lymphocytes. Although such phosphoantigens could prove useful in the composition of vaccines involving gammadelta T cell-mediated immunity, their very low abundance in natural sources limits such applications. Here, we describe the chemical production, purification, and bioactivity of a phosphorylated bromohydrin (BrHPP) analogue that mimics the biological properties of natural phosphoantigens. This compound can be obtained in gram amounts, is easy to detect, and is of high stability in aqueous solutions. Whereas unspecific binding of BrHPP to a wide panel of cell surface receptors is not detected even at micromolar concentrations, nanomolar concentrations specifically trigger effector responses of human gammadelta T lymphocytes. Thus, BrHPP is a novel molecule enabling potent immunostimulation of human gammadelta T lymphocytes.     

Induction of apoptosis and mitosis inhibition by degraded DNA lipotransfection mimicking genotoxic drug effects

Genotoxic damage induces cell cycle arrest and/or apoptosis by activation of p53 oncosuppressor protein. A number of anticancer drugs are genotoxic and their damaging effect upon cells is mediated by this mechanism. Microinjection of defined DNA species directly into nucleus has been reported previously to activate p53 and inhibit cell cycle. Here, we demonstrate that simple addition of heterogeneous degraded DNA to cultured cells (Rat-1 fibroblasts) in combination with lipotransfecting agent DOTAP leads to apoptosis induction and mitosis inhibition by a molecular mechanism which mimics that of the cellular response to genotoxic anticancer agents. Indeed, both cellular effects induced by lipotransfected degraded DNA (essentially, heterogeneous small DNA fragments) are associated to p53 activation and modulated by two apoptosis-related genes, such as bcl-2 and c-myc, which also modulate the apoptotic threshold to anticancer agents. Here we raise the hypothesis of exogenous DNA segment lipotransfection as possible new tool for anticancer therapy.     

Mapping the Naked Neck (NA) and Polydactyly (PO) mutants of the chicken with microsatellite molecular markers

The bulked segregant analysis methodology has been used to map, with microsatellite markers, two morphological mutations in the chicken: polydactyly (PO) and naked neck (NA). These autosomal mutations show partial dominance for NA, and dominance with incomplete penetrance for PO. They were mapped previously to different linkage groups of the classical map, PO to the linkage group IV and NA being linked to the erythrocyte antigen CPPP. An informative family of 70 offspring was produced by mating a sire, heterozygous for each of the mutations, to 7 dams homozygous recessive for each locus. Three DNA pools were prepared, pool PO included 20 chicks exhibiting at least one extra-toe, pool NA included 20 non-polydactyly chicks showing the typical phenotype associated with heterozygosity for the naked neck mutation, and pool NP included 20 chicks exhibiting neither of the mutant phenotypes. Typings were done on an ABI-373 automatic sequencer with 147 microsatellite markers covering most of the genome. An unbalanced distribution of sire marker alleles were detected between pool PO, and pools NA and NP, for two markers of chromosome 2p, MCW0082 and MCW0247. A linkage analysis taking into account the incomplete penetrance of polydactyly (80%) was performed with additional markers of this region and showed that the closest marker to the PO locus was MCW0071 (5 cM, lod score = 9). MCW0071 lies within the engrailed gene EN2 in the chicken. In the mouse, the homologous gene maps on chromosome 5, close to the hemimelic extra-toes mutation Hx. In the case of the NA locus, markers of chromosome 3 were selected because CPPP was mapped on this chromosome. Analysis of individual typings showed a linkage of 5.7 cM (lod score = 13) between the NA locus and ADL0237 in the distal region of chromosome 3q. These results contribute to connecting the former classical map to the molecular genetic map of the chicken, and open the way to the identification of the molecular nature of two developmental mutations of the chicken that are known to occur in many breeds of chickens.     

Biological characterisation of superficial bladder cancer by bivariate cytokeratin 7/DNA analysis, flow cytometric assessment of MIB- 1, and an immunohistochemical study.

A total of 238 cases of bladder carcinoma stages Ta, Tis, T1 were submitted prospectively to multiparameter flow cytometry and immunohistochemical study in order to determine the biological aggressiveness of the tumour. DNA index (DI), S-phase fraction (SPF) obtained by bivariate cytokeratin 7/DNA analyses, and the immunohistochemical evaluation of p53 and MIB-1 were studied in relation to the traditional prognostic factors in bladder cancer (stage and grade). the variance analysis results showed that DNA aneuploidy was significantly associated with high stage (p = 0.0001), high grade (p = 0.0001), high SPF value > or = 5.5% (p = 0.0001), MIB-1 positivity > or = 31% (p = 0.0001) and high expression of p53 (staining involving > 50% of cells, p = 0.0001). Even if there was no statistical significance the hypotetraploid class (1.70 < DI < 1.89) showed poor prognostic biomarkers more frequently than the other aneuploid classes. Out of 238 cases, 101 were also submitted to flow cytometric measurement of MIB-1 (fMIB-1) to study the correlation between cell proliferation and DNA content. Data obtained from fresh, 3:1 methanol/acetone fixed samples were compared with values obtained from both cell cycle analysis methods and routine application of the MIB-1 immunostaining in histological sections. fMIB-1 values were positively correlated with SPF values (r = 0.801, p < 0.01) and S+G2M fraction (percentage of cells in S and in G2M phases) (r = 0.763, p < 0.01) but no correlation with paraffin sections was found. A fMIB-1 value > 7% was strongly associated with aneuploidy (p = 0.0001). The determination of DNA content coupled with the study of the epithelial (cytokeratin 7) and proliferative (MIB-1) markers could be useful in providing important information on the biological behaviour of superficial bladder tumours.     

Reconstitution of the sodium-calcium exchanger from cardiac sarcolemmal vesicles

The Na+-Ca2+ exchanger was extracted from cardiac sarcolemmal vesicles and reconstituted into phospholipid vesicles by a cholate-dialysis method. Reconstitution was attempted with different phospholipids. Phosphatidylcholine alone was ineffective, whereas phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) showed high activity, but a significant Ca2+ uptake in the absence of Na+ gradient. Optimal reconstitution was obtained with a mixture of phosphatidylcholine and phosphatidylserine (9:1, mol/mol). The reconstituted proteoliposomes showed an ouabain-sensitive (Na+ + K+)-ATPase activity and a Na+-Ca2+ exchange with a specific activity comparable to that of the original vesicles. The specificity toward Na+ was also recovered. A partial purification of the exchanger was obtained by the method of transport-specificity fractionation ( Goldin , S.M. and Rhoden , V. (1978) J. Biol. Chem. 253, 2575-2583). When proteoliposomes were reconstituted with sodium oxalate inside and incubated with calcium in the presence of an outwardly directed Na+ gradient, the vesicles containing the Na+-Ca2+ exchanger specifically accumulated calcium which precipitated inside as calcium oxalate. The resulting increase in density allowed separation of the proteoliposomes containing the Na+-Ca2+ exchanger from the rest of the vesicles on a sucrose density gradient.