Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.     

Spectrally Resolved Morphometry of the Nucleus in Hepatocytes Stained by Four Histological Methods

A novel concept of spectrally resolved morphometry for histological specimens was developed using light microscopy combined with spectrally resolved imaging. The spectroscopic characteristics of rat hepatocytes stained by Haematoxylin and Eosin, Romanowsky–Giemsa, periodic acid–Schiff and Masson's trichrome were assessed. Light intensity in the range 450–850 nm was recorded from 10000 pixels of nuclear domains of each stained cell and represented as light transmittance spectra and optical density. In order to identify spectral shifts caused by stain– macromolecule interactions, we compared the spectra of individual stain components with those of DNA and bovine serum albumin. Chromatin and interchromatin areas were classified spectrally using a chosen spectral library followed by morphometric calculations of nuclear domains for each staining method. The spectral fingerprints of Masson's trichrome stain distinguished the nucleolus from the rest of the nuclear c hromatin, enabling the demarcation and calculation of the nucleolar area. Spectrally resolved imaging of human hepatocytes stained by Masson's trichrome stain revealed marked differences between the nucleolar area in normal human hepatocytes compared with hepatocellular carcinoma. Masson's trichrome stain also distinguished the nucleolar area in human breast carcinoma cells and keratinocytes.     

Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica

Major pathogenic functions of Entamoeba histolytica involved in destruction of host tissues are the degradation of extracellular matrix proteins mediated by secreted cysteine proteinases and contact-dependent killing of host cells via membrane-active factors. A soluble protein with an affinity for membranes was purified from amoebic extracts to apparent homogeneity. N-terminal sequencing and subsequent molecular cloning of the factor revealed that it is a member of the cysteine proteinase family of E. histolytica , which we termed CP5. Further experiments with the purified protein showed that it has potent proteolytic activity that is abrogated in the presence of inhibitors specific for cysteine proteinases. The enzyme firmly associates with membranes retaining its proteolytic activity and it produces cytopathic effects on cultured monolayers. A model of the three-dimensional structure of CP5 revealed the presence of a hydrophobic patch that may account for the potential of the protein to associate with membranes. Immunocytochemical localization of the enzyme to the surface of the amoeba in combination with the recent finding that the gene encoding CP5 is missing in the closely related but non-pathogenic Entamoeba dispar suggests a potential role of the protein in host tissue destruction of E. histolytica .     

1,2,5-Trisubstituted 1,4-diazepine-3-one: A novel dipeptidomimetic molecular scaffold

Summary The continuing effort to transform bioactive peptides into non-peptide peptidomimetics of therapeutic potential requires a diversity of tools such as molecular scaffolds, pseudopeptide modifications, and conformation mimetics. To this end, a novel polyfunctional monoheterocyclic system, 1,2,5-trisubstituted hexahydro-3-oxo-1H-1,4-diazepine ring (DAP), was designed. The linear precursor for the DAP was generated through a reductive alkylation step including a modified side chain and an α-amino function of two amino acid derivatives. Structural analysis of model diastereomeric DAPs, employing¹H and¹³C NMR and computer simulation, revealed the conformational preferences of this system. The structural similarities to the 1,4-benzodiazepine, a common molecular scaffold for many non-peptidic peptidomimetic agents, and the pronounced dipeptidomimetic character of the DAP system offer a new powerful tool to medicinal chemists engaged in rational peptide-based drug design.     

Apyrases (nucleoside triphosphate-diphosphohydrolases) play a key role in growth control in Arabidopsis

Expression of two Arabidopsis (Arabidopsis thaliana) apyrase (nucleoside triphosphate-diphosphohydrolase) genes with high similarity, APY1 and APY2, was analyzed during seedling development and under different light treatments using beta-glucuronidase fusion constructs with the promoters of both genes. As evaluated by beta-glucuronidase staining and independently confirmed by other methods, the highest expression of both apyrases was in rapidly growing tissues and/or tissues that accumulate high auxin levels. Red-light treatment of etiolated seedlings suppressed the protein and message level of both apyrases at least as rapidly as it inhibited hypocotyl growth. Adult apy1 and apy2 single mutants had near-normal growth, but apy1apy2 double-knockout plants were dwarf, due primarily to reduced cell elongation. Pollen tubes and etiolated hypocotyls overexpressing an apyrase had faster growth rates than wild-type plants. Growing pollen tubes released ATP into the growth medium and suppression of apyrase activity by antiapyrase antibodies or by inhibitors simultaneously increased medium ATP levels and inhibited pollen tube growth. These results imply that APY1 and APY2, like their homologs in animals, act to reduce the concentration of extracellular nucleotides, and that this function is important for the regulation of growth in Arabidopsis.     

A cell-free system for light-dependent nuclear import of phytochrome

Translocation from the cytosol to the nucleus is an essential step in phytochrome (phy) signal transduction. In the case of phytochrome A (phyA), this step occurs with the help of FHY1 (far-red-elongated hypocotyl 1), a specific transport protein. To investigate the components involved in phyA transport, we used a cell-free system that facilitates the controlled addition of transport factors. For this purpose, we isolated nuclei from the unicellular green algae Acetabularia acetabulum . These nuclei are up to 100 μm in diameter and allow easy detection of imported proteins. Experiments with isolated nuclei of Acetabularia showed that FHY1 is sufficient for phyA transport. The reconstituted system demonstrates all the characteristics of phytochrome transport in Arabidopsis thaliana . In addition, FHY1 was also actively exported from the nucleus, consistent with its role as a shuttle protein in plants. Therefore, we believe that isolated Acetabularia nuclei may be used as a general tool to study nuclear transport of plant proteins.     

Gene expression profiles from formalin fixed paraffin embedded breast cancer tissue are largely comparable to fresh frozen matched tissue

Background and Methods

Formalin Fixed Paraffin Embedded (FFPE) samples represent a valuable resource for cancer research. However, the discovery and development of new cancer biomarkers often requires fresh frozen (FF) samples. Recently, the Whole Genome (WG) DASL (cDNA-mediated Annealing, Selection, extension and Ligation) assay was specifically developed to profile FFPE tissue. However, a thorough comparison of data generated from FFPE RNA and Fresh Frozen (FF) RNA using this platform is lacking. To this end we profiled, in duplicate, 20 FFPE tissues and 20 matched FF tissues and evaluated the concordance of the DASL results from FFPE and matched FF material.

Methodology and Principal Findings

We show that after proper normalization, all FFPE and FF pairs exhibit a high level of similarity (Pearson correlation >0.7), significantly larger than the similarity between non-paired samples. Interestingly, the probes showing the highest correlation had a higher percentage G/C content and were enriched for cell cycle genes. Predictions of gene expression signatures developed on frozen material (Intrinsic subtype, Genomic Grade Index, 70 gene signature) showed a high level of concordance between FFPE and FF matched pairs. Interestingly, predictions based on a 60 gene DASL list (best match with the 70 gene signature) showed very high concordance with the MammaPrint® results.

Conclusions and Significance

We demonstrate that data generated from FFPE material with the DASL assay, if properly processed, are comparable to data extracted from the FF counterpart. Specifically, gene expression profiles for a known set of prognostic genes for a specific disease are highly comparable between two conditions. This opens up the possibility of using both FFPE and FF material in gene expressions analyses, leading to a vast increase in the potential resources available for cancer research.     

The effect of the lunar cycle on fecal cortisol metabolite levels and foraging ecology of nocturnally and diurnally active spiny mice

We studied stress hormones and foraging of nocturnal Acomys cahirinus and diurnal A. russatus in field populations as well as in two field enclosures populated by both species and two field enclosures with individuals of A. russatus alone. When alone, A. russatus individuals become also nocturnally active. We asked whether nocturnally active A. russatus will respond to moon phase and whether this response will be obtained also in diurnally active individuals. We studied giving-up densities (GUDs) in artificial foraging patches and fecal cortisol metabolite levels. Both species exhibited elevated fecal cortisol metabolite levels and foraged to higher GUDs in full moon nights; thus A. russatus retains physiological response and behavioral patterns that correlate with full moon conditions, as can be expected in nocturnal rodents, in spite of its diurnal activity. The endocrinological and behavioral response of this diurnal species to moon phase reflects its evolutionary heritage.     

Intestinal epithelial cells from inflammatory bowel disease patients preferentially stimulate CD4+ T cells to proliferate and secrete interferon-gamma

Previous studies have suggested that intestinal epithelial cells (IECs) have the capacity to function as nonprofessional antigen presenting cells that in the normal state preferentially activate CD8+ T cells. However, under pathological conditions, such as those found in inflammatory bowel disease (IBD), persistent activation of CD4+ T cells is seen. The aim of this study was to determine whether the IBD IECs contribute to CD4+ T cell activation. Freshly isolated human IECs were obtained from surgical specimens of patients with or without IBD and cocultured with autologous or allogeneic peripheral blood T lymphocytes. Cocultures of normal T cells and IECs derived from IBD patients resulted in the preferential activation of CD4+ T cell proliferation that was associated with significant IFN-gamma, but not IL-2, secretion. Cytokine secretion and CD4+ T cell proliferation was inhibited by pretreatment of the IBD IECs with the anti-DR MAb L243. In contrast, normal IECs stimulated the proliferation and cytokine secretion by CD4+ T cells to a significantly lesser degree than IBD IECs. Furthermore, blockade of human leukocyte antigen-DR had a lesser effect in the normal IEC-CD4+ T cell cocultures. We conclude that IECs can contribute to the ongoing CD4+ T cell activation seen in IBD. We suggest that the apparent differences between the secreted levels of IFN-gamma indicate that it may play a dual role in intestinal homeostasis, in which low levels contribute to physiological inflammation whereas higher levels are associated with an uncontrolled inflammatory state.