To study the importance of phosphorylation for p53 transactivation function, we generated mutations at each of its known phosphorylated serine amino acids. Mutations of murine p53 serine residues individually to either alanine or glutamic acid at positions 7, 9, 12, 18, 37, 312, and 389 resulted in equivalent levels of transcriptional activation in standard transient transfection experiments. However, when p53 transcriptional activity was measured in cells that attain G 1 arrest upon contact inhibition, wild-type p53 was inactive, and only alteration at serine 389 to glutamic acid resulted in a functional p53 protein. This Ser 3 Glu mutant also has an increased ability to bind DNA. Elimination of the phosphorylation site by substitution of an alanine amino acid resulted in loss of transcriptional activity. We also demonstrated that specific phosphorylation of p53 at serine 389 is induced by cyclin E overexpression in high-density cells. Our data establish for the first time that phosphorylation of p53 at serine 389 is important in activating its function in vivo.The p53 tumor suppressor is a transcriptional activator that regulates several cellular processes including cell cycle control, apoptosis, and DNA repair (1, 2). The wild-type p53 protein has been shown to bind to specific DNA sequences in the promoters or introns of several genes, including p21 WAF1/cip1 , mdm2, gadd45, bax, and cyclin G, activating their transcription (3-8).The majority of inactivating mutations in the p53 gene disrupt the DNA-binding domain and hence the transcriptional activation function of p53 (9, 10).The role of p53 in the cell cycle has been characterized in some detail. Activation of the p21 WAF1/cip1 gene by p53 results in growth suppression of cultured cells (4). p21WAF1/cip1 was also independently identified as an inhibitor of the cell cycle regulators cyclin/cyclin-dependent kinases (Cdk) 1 (11). p21 WAF1/cip1 seems to be a universal inhibitor of the cyclin-Cdk complexes, although it inhibits the cyclin E-Cdk2 complex most potently (11). The binding of cyclin E to Cdk2 activates its kinase function at the G 1 /S stage of the cell cycle (12, 13).The p53 protein is phosphorylated at several serine amino acids. A cluster of phosphoserines has been identified in vivo in the transactivation domain of murine p53 at amino acids 7, 9, 18, and 37 (14). In vitro studies showed that purified casein kinase I can phosphorylate serines 7, 9, and 12 (15). Purified DNA-activated protein kinase has also been shown to phosphorylate serines 7 and 18 in murine p53 and serines 15 and 37 in human p53 (16). Experiments examining the role of p53 phosphorylated amino-terminal amino acids in transcriptional activation indicated that mutations of these serines did not alter p53 function (17,18). Other data indicated a weak effect of phosphorylation at serine 15 (19, 20). Thus, the phosphorylation of amino-terminal serines has little if any effect on p53 function.Two phosphoserines in the C-terminal domain, serines 312 and 389 in murine p53, have been...

The wild-type p53 protein functions to suppress

eThe recent reports of resistance in Plasmodium falciparum to artemisinin derivatives and their partner drugs demand intensive studies toward understanding the molecular mechanisms of resistance. In this study, we examined the in vitro susceptibility of 63 P. falciparum field isolates collected from the China-Myanmar border area to chloroquine (CQ) and piperaquine (PPQ). Parasite isolates remained highly resistant to CQ, with the geometric mean 50% inhibitory concentration (IC 50 ) of 252.7 nM and a range of 51.9 to 1,052.0 nM. In comparison, these parasites had a geometric mean IC 50 of 28.4 nM for PPQ, with a fairly wide range of 5.3 to 132.0 nM, suggesting that certain parasite isolates displayed relatively high levels of resistance to PPQ. Interestingly, within the 4 years of study, the parasites exhibited a continuous decline in susceptibilities to both CQ and PPQ, and there was a significant correlation between responses to CQ and PPQ (Pearson correlation coefficient ‫؍‬ 0.79, P < 0.0001). Consistent with the CQ-resistant phenotype, all parasites carried the pfcrt K76T mutation, and most parasites had the CVIET type that is prevalent in Southeast Asia. In contrast, pfmdr1 mutations were relatively rare, and no gene amplification was detected. Only the pfmdr1 N1042D mutation was associated with resistance to CQ. For the pfmrp1 gene, four substitutions reached relatively high prevalence of >22%, and the I876V mutation was associated with reduced sensitivity to CQ. However, we could not establish a link between PPQ responses and the polymorphisms in the three genes associated with quinoline drug resistance.

The wild-type p53 protein is a potent growth suppressor when overexpressed in vitro. It functions as a transcriptional activator and causes growth arrest at the G1/S stage of the cell cycle. We monitored p53 transactivation as an indicator of p53 function throughout the cell cycle. We first demonstrate that cells which exhibited contact inhibition of growth lacked p53 transactivation function at high cell density. Since these cells were noncycling, we examined whether the ectopic expression of any cyclin could override contact inhibition of growth and restore p53 transactivation function. The transfection of cyclin E at high cell density stimulated the progression of cells through the cell cycle and restored p53 transactivation function. The transcriptional activity of p53 induced by cyclin E was regulated at the level of DNA binding. Cells that did not show contact inhibition of growth had a functional p53 regardless of cell density. Thus, contact inhibition of cell growth corresponded to a lack of p53 transactivation function and the overexpression of cyclin E in these contact-inhibited cells stimulated cell cycle progression and resulted in p53 transcriptional activity.

We evaluated the dynamics of parasite populations during in vitro culture adaptation in 15 mixed Plasmodium falciparum infections, which were collected from a hypoendemic area near the China-Myanmar border. Allele types at the msp1 block 2 in the initial clinical samples and during subsequent culture were quantified weekly using a quantitative PCR method. All mixed infections carried two allele types based on the msp1 genotyping result. We also genotyped several polymorphic sites in the dhfr, dhps and mdr1 genes on day 0 and day 28, which showed that most of the common sites analyzed were monomorphic. Two of the three clinical samples mixed at dhps 581 remained stable while one changed to wild-type during the culture. During in vitro culture, we observed a gradual loss of parasite populations with 10 of the 15 mixed infections becoming monoclonal by day 28 based on the msp1 allele type. In most cases, the more abundant msp1 allele types in the clinical blood samples at the beginning of culture became the sole or predominant allele types on day 28. These results suggest that some parasites may have growth advantages and the loss of parasite populations during culture adaptation of mixed infections may lead to biased results when comparing the phenotypes such as drug sensitivity of the culture-adapted parasites.

Plasmodium falciparum is usually asynchronous during in vitro culture. Highly synchronized cultures of Plasmodium falciparum are routinely used in malaria research. Here, we describe a simple synchronization procedure for P. falciparum asexual erythrocytic culture, which involves storage at 4°C for 8–24 h followed by routine culture. When cultures with 27–60% of ring stage were synchronized using this procedure, 70–93% ring stages were obtained after 48 h of culture and relative growth synchrony remained for at least two erythrocytic cycles. To test the suitability of this procedure for subsequent work, drug sensitivity assays were performed using four laboratory strains and four freshly adapted clinical P. falciparum isolates. Parasites synchronized by sorbitol treatment or refrigeration showed similar dose-response curves and comparable IC50 values to four antimalarial drugs. The refrigeration synchronization method is simple, inexpensive, time-saving, and should be especially useful when large numbers of P. falciparum culture are handled.

The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.