Trafficking of the phosphoprotein PfCRT to the digestive vacuolar membrane in Plasmodium falciparum.

The digestive vacuole plays an important role in the pathophysiology of the human malaria parasite Plasmodium falciparum. It is a terminal degradation organelle involved in the proteolysis of the host erythrocyte's haemoglobin; it is the site of action of several antimalarial drugs and its membrane harbours transporters implicated in drug resistance. How the digestive vacuole recruits residential proteins is largely unknown. Here, we have investigated the mechanism underpinning trafficking of the chloroquine resistance transporter, PfCRT, to the digestive vacuolar membrane. Nested deletion analysis and site-directed mutagenesis identified threonine 416 as a functional residue for sorting PfCRT to its site of residence. Mass spectroscopy demonstrated that threonine 416 can be phosphorylated. Further phosphorylation was detected at serine 411. Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane.

Association of socio-economic status and stage of pancreatic cancer at time of surgery in a German setting.

BACKGROUND/AIMS: Curative resection has been proven to be one of the most important factors determining outcome in pancreatic cancer patients. Advanced stage of pancreatic cancer at diagnosis is strongly associated with a low socioeconomic status (SES), and patients from affluent areas have better cancer survival than patients from deprived areas. We tested, in our population of pancreatic cancer patients, the hypothesis that surrogates representing a lower SES or demographic factors (DGF) linked to rural areas are associated with a more advanced disease stage at presentation. METHODOLOGY: Between 1989 and 2008, patients with pancreatic adenocarcinoma and pancreaticoduodenectomy were identified from our pancreatic resection database. DGF, SES surrogates and tumor stage were obtained from patients' files together with pathology reports, a residents' registration office questionnaire and telephone interviews with patients and family members. RESULTS: Follow-up was completed in 117 patients. There were no significant differences regarding tumor stage (local size and lymph node metastases), or the likelihood of negative resection margins in relation to the patients' DGF or any surrogate parameters for SES. Furthermore, comparison of two different treatment periods showed no significant advances regarding secondary cancer prevention within 20 years. CONCLUSIONS: Longer waiting times for appointments combined with less sensitive imaging techniques and consecutive later referral to a cancer specialist are likely to be associated with inferior quality of medical results. Therefore, a lively debate is currently underway in Germany concerning the harmonization of reimbursement modes for statutory and private health insurance. Our data with no negative correlation of low SES or unfavorable DGF and disease stage at time of presentation or the likelihood for a curative resection, do not promote the universal accusation of health care disparities solely based on economic issues in Germany.

Quantitative pH measurements in Plasmodium falciparum-infected erythrocytes using pHluorin.

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of action of several antimalarial drugs, such as chloroquine, which accumulate in this organelle due to their properties as amphiphilic weak bases that inhibit haem detoxification. It has been suggested that changes in the pH of the digestive vacuole, affecting either drug partitioning or haem solubility and/or biomineralization rates, would correlate with reduced intracellular chloroquine accumulation and, hence, would determine the chloroquine-resistance phenotype. The techniques previously used to quantify digestive vacuolar pH mainly relied on lysed or isolated parasites, with unpredictable consequences on internal pH homeostasis. In this study, we have investigated the baseline steady-state pH of the cytoplasm and digestive vacuole of a chloroquine-sensitive (HB3) and a chloroquine-resistant (Dd2) parasite using a pH-sensitive green fluorescent protein, termed pHluorin. This non-invasive technique allows for in vivo pH measurements in intact P. falciparum-infected erythrocytes under physiological conditions. The data suggest that the pH of the cytoplasm is approximately 7.15 +/- 0.07 and that of the digestive vacuole approximately 5.18 +/- 0.05. No significant differences in baseline pH values were recorded for the chloroquine-sensitive and chloroquine-resistant parasites.

Structure and regulation of the murine Mash2 gene.

Transcription factors of the basic helix-loop-helix family such as Mash2 are essential for adequate differentiation of the trophoblast. Disruption of the Mash2 gene leads to early intrauterine death caused by placental insufficiency with an absent spongiotrophoblast and an underdeveloped chorion. The aim of the present study was to analyze the structure of the murine Mash2 gene, to screen a broad spectrum of organs for its expression, and to investigate placental Mash2 expression at different gestational ages. The RNase protection assay identified, in addition to the postulated Mash2 mRNA, two unexpected Mash2 transcripts that could be confirmed by a 5' rapid amplification of cDNA ends. However, all three transcripts were detectable exclusively in murine placenta and not in other organs, such as the ovary, uterus, skin, lung, femur, skeletal muscle, kidney, skull, adrenal gland, tongue, stomach, spleen, skin, testis, or pancreas. Sequence analysis disclosed an additional transcription start site upstream of exon 2. Placental Mash2 mRNA is measurable at all investigated stages of gestation. After its initial detection on Day 8.5 postcoitum (p.c.; set to 100%; 100.0% +/- 28.4%), the Mash2 mRNA concentration increases significantly and reaches a maximum of 812.0% +/- 69.7% on Day 12.5 p.c. The second half of gestation is marked by a more than 8-fold Mash2 decrease by Day 18.5 p.c. (77.0% +/- 28.4%). A 36.9% +/- 4.7% level of placental Mash2 mRNA is measurable at term.