On the enigmatic disappearance of Rauber's layer.

The polar trophoblast overlays the epiblast in eutherian mammals and, depending on the species, has one of two different fates. It either remains a single-layered, thinning epithelium called "Rauber's layer," which soon disintegrates, or, alternatively, it keeps proliferating, contributing heavily to the population of differentiating, invasive trophoblast cells and, at least in mice, to the induction of gastrulation. While loss of the persistent polar trophoblast in mice leads to reduced induction of gastrulation, we show here that prevention of the loss of the polar trophoblast in cattle results in ectopic domains of the gastrulation marker, BRACHYURY This phenotype, and increased epiblast proliferation, arose when Rauber's layer was maintained for a day longer by countering apoptosis through BCL2 overexpression. This suggests that the disappearance of Rauber's layer is a necessity, presumably to avoid excessive signaling interactions between this layer and the subjacent epiblast. We note that, in all species in which the polar trophoblast persists, including humans and mice, ectopic polar trophoblast signaling is prevented via epiblast cavitation which leads to the (pro)amniotic cavity, whose function is to distance the central epiblast from such signaling interactions.

Proteomic phenotyping of Novosphingobium nitrogenifigens reveals a robust capacity for simultaneous nitrogen fixation, polyhydroxyalkanoate production, and resistance to reactive oxygen species.

Novosphingobium nitrogenifigens Y88(T) (Y88) is a free-living, diazotrophic Alphaproteobacterium, capable of producing 80% of its biomass as the biopolymer polyhydroxybutyrate (PHB). We explored the potential utility of this species as a polyhydroxybutyrate production strain, correlating the effects of glucose, nitrogen availability, dissolved oxygen concentration, and extracellular pH with polyhydroxybutyrate production and changes in the Y88 proteomic profile. Using two-dimensional differential in-gel electrophoresis and tandem mass spectrometry, we identified 217 unique proteins from six growth conditions. We observed reproducible, characteristic proteomic signatures for each of the physiological states we examined. We identified proteins that changed in abundance in correlation with either nitrogen fixation, dissolved oxygen concentration, or acidification of the growth medium. The proteins that correlated with nitrogen fixation were identified either as known nitrogen fixation proteins or as novel proteins that we predict play roles in aspects of nitrogen fixation based on their proteomic profiles. In contrast, the proteins involved in central carbon and polyhydroxybutyrate metabolism were constitutively abundant, consistent with the constitutive polyhydroxybutyrate production that we observed in this species. Three proteins with roles in detoxification of reactive oxygen species were identified in this obligate aerobe. The most abundant protein in all experiments was a polyhydroxyalkanoate granule-associated protein, phasin. The full-length isoform of this protein has a long, intrinsically disordered Ala/Pro/Lys-rich N-terminal segment, a feature that appears to be unique to sphingomonad phasins. The data suggest that Y88 has potential as a PHB production strain due to its aerobic tolerance and metabolic orientation toward polyhydroxybutyrate accumulation, even in low-nitrogen growth medium.

Paclitaxel effects on the proteome of HL-60 promyelocytic leukemic cells: comparison to peloruside A.

Paclitaxel (Taxol®), a drug used to treat solid tumors of the breast, ovary and lung, stabilizes microtubules and arrests cells in G(2)/M of the cell cycle. Using two-dimensional differential in-gel electrophoresis (DIGE), we examined the proteomic response of a human HL-60 promyeloid leukemic cell line to paclitaxel. Our intention was to compare the effects of paclitaxel to those of a new-generation microtubule-stabilizing agent, peloruside A, investigated in an earlier study. In response to 100 nM paclitaxel treatment for 24 h, 21 identified proteins changed in abundance, with 13 increases and 8 decreases. In addition, 21 other unidentified proteins were also changed by treatment with paclitaxel. Using Western blotting, the transcription factor c-Myc was shown to be reduced in abundance by both drugs. Our results showed both differences and similarities at the single protein level between paclitaxel and peloruside A, although the same general classes of proteins: cytoskeletal, nucleic acid binding, stress, and apoptotic proteins, changed following exposure. The proteomic response to paclitaxel was more extensive than the response to an equipotent dose of peloruside A.

Acquired resistance to peloruside A and laulimalide is associated with downregulation of vimentin in human ovarian carcinoma cells.

PURPOSE: Acquired ß-tubulin alterations in human ovarian carcinoma 1A9 cells were previously shown to confer resistance to the microtubule stabilizing agents peloruside A (PLA) and laulimalide (LAU). We examined the proteome of resistant cells to see what other protein changes occurred as a result of the acquired drug resistance. METHODS: Two-dimensional differential in-gel electrophoresis was performed to explore differentially expressed proteins in the resistant 1A9-R1 (R1) and 1A9-L4 (L4) cells. The proteins on the gels were identified by MALDI-TOF MS, and altered protein abundance was confirmed by Western blotting and immunocytochemistry. Vimentin expression was restored in vimentin-deficient L4 cells by transfecting a full-length human vimentin cDNA, and sensitivity to PLA and LAU were tested using an MTT cell proliferation assay. RESULTS: Proteomic analysis identified several proteins that were significantly altered in the resistant cells relative to the parental 1A9 cells. Using Western blotting and immunocytochemistry, a decreased vimentin abundance in the L4 cells was validated. Vimentin levels were unchanged in PLA-resistant R1 cells and paclitaxel/epothilone-resistant derivatives of 1A9 cells. Vimentin cDNA transfection into L4 cells partially restored PLA and LAU sensitivity. CONCLUSIONS: Downregulation of vimentin contributes to the resistance of 1A9 cells to the microtubule stabilizing agents, PLA and LAU.

Identification of cytokeratin 18 as a biomarker of mouse and human hepatosplenic schistosomiasis.

Previously, we demonstrated unique protein expression patterns in 20-week-Schistosoma mansoni-infected CBA/J mice with moderate splenomegaly syndrome (MSS) or hypersplemomegaly syndrome (HSS). To better understand the development of severe pathology, we compared the two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic signatures of livers from uninfected mice and mice infected for 6, 8, 12, or 20 weeks and found significant changes in collagen isoforms, interleukin-2 (IL-2), cytokeratin 18, hydroxyproline, S. mansoni phosphoenolpyruvate carboxykinase, major urinary protein isoforms, and peroxiredoxin 6. Cytokeratin 18, hydroxyproline, and connective tissue growth factor (CTGF) were chosen for analysis in mouse and human sera using targeted biochemical assays. Consistent with the liver analysis, cytokeratin 18, CTGF, and hydroxyproline were significantly elevated in sera from mice with HSS compared to those from uninfected mice or mice with MSS. Moreover, cytokeratin 18 and CTGF were found to be markers for subjects with hepatosplenic and intestinal schistosomiasis, respectively, while serum hydroxyproline was a strong indicator of fibrosis for severe HS. These findings indicate that schistosome-associated changes to the liver can be detected in the serum and reveal the potential for cytokeratin 18 to be used as a diagnostic marker for early detection of hepatosplenic schistosomiasis.

Effects of the microtubule stabilizing agent peloruside A on the proteome of HL-60 cells.

Peloruside A, isolated from the marine sponge Mycale hentscheli, has a similar mechanism of action to paclitaxel (Taxol®), a drug used clinically to treat tumors of the breast, ovary and lung. Paclitaxel and peloruside stabilize the polymerized form of tubulin and arrest cells in G2/M of the cell cycle. We have therefore used two-dimensional electrophoresis of proteins to examine the effect of peloruside A on the human HL-60 promyeloid leukemic cell line. Our goals included investigation whether affected proteins could be mapped onto pathways that predicted the cellular effects of this compound. In response to 100 nM peloruside A treatment for 24 h, seventeen identified proteins showed significant change in abundance with fourteen increases and three decreases. Use of Ingenuity Pathways Analysis confirmed that peloruside A affected pathways consistent with the known effects on microtubules and apoptosis. Our results also indicate a potential role of c-Myc in the cellular actions of peloruside consistent with an induction of aneuploidy seen at low concentrations of peloruside.

The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate-washed membrane standard.

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress.

Compatibility of toluidine blue with laser microdissection and saturation labeling DIGE.

Tissue fixation and staining protocols for laser microdissection are frequently not fully compatible with subsequent proteomic analysis. We compared the effect of three common histological stains (toluidine blue (TB), hemotoxylin, and hematoxylin and eosin (HE)) on tissue visualization, protein recovery, the saturation labeling reaction, and 2-D electrophoresis. TB provided the best visualization of colorectal tumor tissue during laser microdissection (LMD) and had a comparable effect on protein recovery and the saturation labeling reaction with hematoxylin, provided a modified 2-D clean-up protocol was used. Eosin inhibited both protein recovery and the saturation labeling reaction.

Profiling the metabolic proteome of bovine mammary tissue.

2-DE and MALDI mass fingerprinting were used to analyse mammary tissue from lactating Friesian cows. The goal was detection of enzymes in metabolic pathways for synthesis of milk molecules including fatty acids and lactose. Of 418 protein spots analysed by PMF, 328 were matched to database sequences, resulting in 215 unique proteins. We detected 11 out of the 15 enzymes in the direct pathways for conversion of glucose to fatty acids, two of the pentose phosphate pathway enzymes and two of the enzymes for lactose synthesis from glucose. We did not detect enzymes that catalyse the first three reactions of glycolysis. Our results are typical of enzyme detection using 2-DE of mammalian tissues. We therefore advocate caution when relating enzyme abundances measured by 2-DE to metabolic output as not all relevant proteins are detected. 2-D DIGE was used to measure interindividual variation in enzyme abundance from eight animals. We extracted relative protein abundances from 2-D DIGE data and used a logratio transformation that is appropriate for compositional data of the kind represented in many proteomics experiments. Coefficients of variation for abundances of detected enzymes were 3-8%. We recommend use of this transformation for DIGE and other compositional data.

Autologous dendritic cells pulsed with eluted peptide as immunotherapy for advanced B-cell malignancies.

We have studied the feasibility, safety and efficacy of vaccination with autologous dendritic cells pulsed with eluted peptide in patients with advanced low-grade B-cell malignancies. This study demonstrates that autologous dendritic cell vaccines can be successfully produced from patients with advanced disease and be delivered without significant toxicity. Furthermore, we have demonstrated immunological and clinical responses in two of ten patients treated. These results provide further evidence for the use of immunotherapy in the management of B-cell malignancies, but also suggest that sustained responses may only be possible in patients with low bulk disease early in the disease course.

Phenotypic population screen identifies a new mutation in bovine DGAT1 responsible for unsaturated milk fat.

Selective breeding has strongly reduced the genetic diversity in livestock species, and contemporary breeding practices exclude potentially beneficial rare genetic variation from the future gene pool. Here we test whether important traits arising by new mutations can be identified and rescued in highly selected populations. We screened milks from 2.5 million cows to identify an exceptional individual which produced milk with reduced saturated fat content, and improved unsaturated and omega-3 fatty acid concentrations. The milk traits were transmitted dominantly to her offspring, and genetic mapping and genome sequencing revealed a new mutation in a previously unknown splice enhancer of the DGAT1 gene. Homozygous carriers show features of human diarrheal disorders, and may be useful for the development of therapeutic strategies. Our study demonstrates that high-throughput phenotypic screening can uncover rich genetic diversity even in inbred populations, and introduces a novel strategy to develop novel milks with improved nutritional properties.

Metabolic proteomics of the liver and mammary gland during lactation.

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by ß-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and ß-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than ß-oxidation.

Peloruside- and laulimalide-resistant human ovarian carcinoma cells have ßI-tubulin mutations and altered expression of ßII- and ßIII-tubulin isotypes.

Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on ß-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the ßI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a ß-tubulin-binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the ß-tubulin isotype composition of the cells was examined. Increased expression of ßII- and ßIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action.

Proteomic analysis of microsomes from lactating bovine mammary gland.

Mammary gland has multiple metabolic potential including for large-scale synthesis of milk proteins, carbohydrate, and lipids including nutrient triacylglycerols. We have carried out a proteomic analysis of mammary tissue to discover proteins that affect lipid metabolism. Unfractionated microsomes from lactating bovine mammary tissue were analyzed using one-dimensional SDS-PAGE with RPLC-ESI-MS/MS. This approach gave 703 proteins including 160 predicted transmembrane proteins. Proteins were classified according to their subcellular localizations and biological functions. Over 50 proteins were associated with cellular uptake, metabolism, and secretion of lipids, including some enzymes that have been previously associated with breast cancer and potential therapeutic targets. This database develops a proteomic view of the metabolic potential of mammary gland that can be expected to contribute to a greater understanding of gene expression and tissue remodeling associated with lactation, and to further dissection of normal and pathological processes in mammary tissue.

Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection.

We found that the proteome of apoptotic T cells includes prominent fragments of cellular proteins generated by caspases and that a high proportion of distinct T cell epitopes in these fragments is recognized by CD8+ T cells during HIV infection. The frequencies of effector CD8+ T cells that are specific for apoptosis-dependent epitopes correlate with the frequency of circulating apoptotic CD4+ T cells in HIV-1-infected individuals. We propose that these self-reactive effector CD8+ T cells may contribute to the systemic immune activation during chronic HIV infection. The caspase-dependent cleavage of proteins associated with apoptotic cells has a key role in the induction of self-reactive CD8+ T cell responses, as the caspase-cleaved fragments are efficiently targeted to the processing machinery and are cross-presented by dendritic cells. These findings demonstrate a previously undescribed role for caspases in immunopathology.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: the role of the virus.

In a companion study, we showed a dichotomy between the expansion of central memory (CCR7(+)) hepatitis C virus (HCV)-specific CTL and the incomplete memory effector differentiation in patients with acute HCV infection. Indeed, effector cells were unable to perform immediate functions, despite expressing the tissue-homing phenotype of effector memory cells (CCR7(-); semi-effectors). However, since they promptly differentiated into full-effectors upon IL-2 contact, we suggested that the inhibitory effect by environmental (possibly viral) factors on IL-2 production may have a pivotal role in generating the large population of semi-effector CCR7(-)/IFN-gamma(-) CTL. In accord with this view, we report here strong evidence in support of circulating HCVcore protein (HCVcore) playing a central role in inhibiting effector CTL differentiation, but not memory CTL expansion. The regulatory HCVcore effect is related to inhibition of the signal transduction pathway instrumental for IL-2 production, supporting the evidence that IL-2 was capable both of pushing semi-effector CTL to complete their effector cell program and of restoring the HCVcore-dependent inhibitory effect. Therefore, the strength of CTL activation is dependent on the balance between the threshold of stimulatory signals and the viral interference capacities provided during priming.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms.

Hallmark of acute hepatitis C virus (HCV) infection is a severe virus-specific effector CD8(+) T cell dysfunction that seems to be a critical factor in preventing the resolution of infection and in favoring the onset of chronic liver immunopathology. We suggest that this dysfunction is critical in the establishment of HCV persistence, unless it is compensated by multispecific responses, as found in individuals resolving infection. Analyses on purified populations indicate that central memory HCV-specific CCR7(+)/CD8(+) T cells efficiently proliferate and differentiate in vitro, although the large population of memory effector CCR7(-) cells found in the peripheral blood of acutely infected patients display poor effector functions ex vivo (semi-effectors). However, we report strong evidence in support of IL-2 being capable of pushing semi-effector CTL to complete their effector cell program. Therefore, IL-2 deficiency during T cell activation may be responsible for the dichotomy between memory CTL expansion and incomplete effector differentiation shown in patients with acute HCV infection. These data are consistent with the possible therapeutic treatment with IL-2 to rebuild the effector T cell pool in these patients.