A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.     

Structural,dynamic and mechanical properties of POPC at low cholesterol concentration studied in pressure/temperature space

We have studied the structural, dynamic and mechanical properties of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine (POPC)/cholesterol binary mixtures by small-angle X-ray scattering. Our investigations were concentrated on the biologically most relevant pressure-temperature-cholesterol regime, i.e. the liquid crystalline phase and its phase boundary to the lamellar gel phase within a cholesterol concentration up to 25 mol%. From the dependence of the transition pressure we derived a value of 19 kJ/mol for the transition enthalpy Delta H(m) of POPC in excess water. With increasing cholesterol concentration, Delta H(m) drops to about 7 kJ/mol at 20 mol% cholesterol. Time-resolved pressure-scan (p-scan) and temperature-jump (T-jump) experiments reveal that at low cholesterol content (<5-8 mol%) the fluidity and also the bilayer compressibility increase remarkably. In contrast, at concentrations between 5 and 25 mol% cholesterol the bilayer becomes again more rigid and the lipid bilayer spacing increases about 2 A. Theses changes are attributed to the onset of phase separation between liquid disordered and liquid ordered phases. The fluid-fluid miscibility gap for this mono-unsaturated lecithin species is strongly enlarged compared with saturated lecithins.     

Pluripotency deficit in clones overcome by clone-clone aggregation: epigenetic complementation?

Abnormal gene expression patterns in somatic cell clones and their attrition in utero are commonly considered a consequence of errors in nuclear reprogramming. We observe that mouse clone blastocysts have less than half the normal cell number, and that higher cell number correlates with correct expression of Oct4, a gene essential for peri-implantation development and embryonic pluripotency. To increase the cell number, we aggregated genetically identical clones at the 4-cell stage. Clone-clone aggregates did not form more blastocysts, but the majority expressed Oct4 normally and had higher rates of fetal and postnatal development. Fertilized blastocysts with low cell numbers, induced by removal of two blastomeres at the 4-cell stage, did not exhibit abnormal Oct4 expression, indicating that improved gene expression and post-implantation development of clone-clone aggregates is not a consequence of increased cell number. Rather, we propose that complementation of non-cell-autonomous defects of genetically identical, but epigenetically different, embryos results in improved gene expression in clone-clone aggregates.     

Disruption of the pelota gene causes early embryonic lethality and defects in cell cycle progression

Mutations in either the Drosophila melanogaster pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast. These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle. To determine the role of the pelota in mouse development and progression of cell cycle, we have established a targeted disruption of the mouse PELO: Heterozygous animals are variable and fertile. Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo(-/-) pups and suggests an embryo-lethal phenotype. Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5). The failure of mitotic active inner cell mass of the Pelo(-/-) blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation. Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo(-/-) embryos at E7.5. Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability.     

The Patient- And Nutrition-Derived Outcome Risk Assessment Score (PANDORA): Development of a Simple Predictive Risk Score for 30-Day In-Hospital Mortality Based on Demographics,Clinical Observation,and Nutrition

ObjectiveTo develop a simple scoring system to predict 30 day in-hospital mortality of in-patients excluding those from intensive care units based on easily obtainable demographic, disease and nutrition related patient data.MethodsScore development with general estimation equation methodology and model selection by P-value thresholding based on a cross-sectional sample of 52 risk indicators with 123 item classes collected with questionnaires and stored in an multilingual online database.SettingWorldwide prospective cross-sectional cohort with 30 day in-hospital mortality from the nutritionDay 2006-2009 and an external validation sample from 2012.ResultsWe included 43894 patients from 2480 units in 32 countries. 1631(3.72%) patients died within 30 days in hospital. The Patient- And Nutrition-Derived Outcome Risk Assessment (PANDORA) score predicts 30-day hospital mortality based on 7 indicators with 31 item classes on a scale from 0 to 75 points. The indicators are age (0 to 17 points), nutrient intake on nutritionDay (0 to 12 points), mobility (0 to 11 points), fluid status (0 to 10 points), BMI (0 to 9 points), cancer (9 points) and main patient group (0 to 7 points). An appropriate model fit has been achieved. The area under the receiver operating characteristic curve for mortality prediction was 0.82 in the development sample and 0.79 in the external validation sample.ConclusionsThe PANDORA score is a simple, robust scoring system for a general population of hospitalised patients to be used for risk stratification and benchmarking.     

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.     

Identification of Genes Whose Expression Profile Is Associated with Non-Progression towards AIDS Using eQTLs

Background

Many genome-wide association studies have been performed on progression towards the acquired immune deficiency syndrome (AIDS) and they mainly identified associations within the HLA loci. In this study, we demonstrate that the integration of biological information, namely gene expression data, can enhance the sensitivity of genetic studies to unravel new genetic associations relevant to AIDS.

Methods

We collated the biological information compiled from three databases of expression quantitative trait loci (eQTLs) involved in cells of the immune system. We derived a list of single nucleotide polymorphisms (SNPs) that are functional in that they correlate with differential expression of genes in at least two of the databases. We tested the association of those SNPs with AIDS progression in two cohorts, GRIV and ACS. Tests on permuted phenotypes of the GRIV and ACS cohorts or on randomised sets of equivalent SNPs allowed us to assess the statistical robustness of this method and to estimate the true positive rate.

Results

Eight genes were identified with high confidence (p = 0.001, rate of true positives 75%). Some of those genes had previously been linked with HIV infection. Notably, ENTPD4 belongs to the same family as CD39, whose expression has already been associated with AIDS progression; while DNAJB12 is part of the HSP90 pathway, which is involved in the control of HIV latency. Our study also drew our attention to lesser-known functions such as mitochondrial ribosomal proteins and a zinc finger protein, ZFP57, which could be central to the effectiveness of HIV infection. Interestingly, for six out of those eight genes, down-regulation is associated with non-progression, which makes them appealing targets to develop drugs against HIV.     

Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III

Dipeptidyl peptidases III (DPPs III) form a distinct metallopeptidase family characterized by the unique HEXXGH motif. High susceptibility to inactivation by organomercurials suggests the presence of a reactive cysteine residue(s) in, or close to, their active site. Yeast DPP III contains five Cys, none of which is absolutely conserved within the family. In order to identify reactive residue(s), site-directed mutagenesis on yeast His₆-tagged DPP III was employed to substitute specifically all five cysteine residues to serine. The variant enzymes thus obtained were enzymatically active and showed an overall structure not greatly affected by the mutations as judged by circular dichroism. Analysis by native and SDS-PAGE under non-reducing conditions revealed the existence of a monomeric and dimeric form in all DPP III proteins except in the C130S, implying that dimerization of yeast DPP III is mediated by the surface-exposed cysteine 130.     

Proteomic changes in maize roots after short-term adjustment to saline growth conditions

It is of fundamental importance to understand adaptation processes leading to salt resistance. The initial effects on maize roots in the first hour after the adjustment to saline conditions were monitored to elucidate initial responses. The subsequent proteome change was monitored using a 2‐D proteomic approach. We found several new salt‐inducible proteins, whose expression has not been previously reported to be modulated by salt. A set of phosphoproteins in maize was detected but only ten proteins were phosphorylated and six proteins were dephosphorylated after the application of 25 mM NaCl for 1 h. Some of the phosphorylated maize proteins such as fructokinase, UDP‐glucosyl transferase BX9, and 2‐Cys‐peroxyredoxine were enhanced, whereas an isocitrate‐dehydrogenase, calmodulin, maturase, and a 40‐S‐ribosomal protein were dephosphorylated after adjustment to saline conditions. The initial reaction of the proteome and phosphoproteome of maize after adjustment to saline conditions reveals members of sugar signalling and cell signalling pathways such as calmodulin, and gave hint to a transduction chain which is involved in NaCl‐induced signalling. An alteration of 14‐3‐3 proteins as detected may change plasma membrane ATPase activity and cell wall growth regulators such as xyloglucane endotransglycosylase were also found to be changed immediately after the adjustment to salt stress.