Identification of Small Molecules That Suppress Ricin-Induced Stress-Activated Signaling Pathways

Ricin is a member of the ribosome-inactivating protein (RIP) family of plant and bacterial toxins. In this study we used a high-throughput, cell-based assay to screen more than 118,000 compounds from diverse chemical libraries for molecules that reduced ricin-induced cell death. We describe three compounds, PW66, PW69, and PW72 that at micromolar concentrations significantly delayed ricin-induced cell death. None of the compounds had any demonstrable effect on ricin''s ability to arrest protein synthesis in cells or on ricin''s enzymatic activity as assessed in vitro. Instead, all three compounds appear to function by blocking downstream stress-induced signaling pathways associated with the toxin-mediated apoptosis. PW66 virtually eliminated ricin-induced TNF-α secretion by J774A.1 macrophages and concomitantly blocked activation of the p38 MAPK and JNK signaling pathways. PW72 suppressed ricin-induced TNF-α secretion, but not p38 MAPK and JNK signaling. PW69 suppressed activity of the executioner caspases 3/7 in ricin toxin- and Shiga toxin 2-treated cells. While the actual molecular targets of the three compounds have yet to be identified, these data nevertheless underscore the potential of small molecules to down-regulate inflammatory signaling pathways associated with exposure to the RIP family of toxins.     

Identification of MiR-205 As a MicroRNA That Is Highly Expressed in Medullary Thymic Epithelial Cells

Thymic epithelial cells (TECs) support T cell development in the thymus. Cortical thymic epithelial cells (cTECs) facilitate positive selection of developing thymocytes whereas medullary thymic epithelial cells (mTECs) facilitate the deletion of self-reactive thymocytes in order to prevent autoimmunity. The mTEC compartment is highly dynamic with continuous maturation and turnover, but the genetic regulation of these processes remains poorly understood. MicroRNAs (miRNAs) are important regulators of TEC genetic programs since miRNA-deficient TECs are severely defective. However, the individual miRNAs important for TEC maintenance and function and their mechanisms of action remain unknown. Here, we demonstrate that miR-205 is highly and preferentially expressed in mTECs during both thymic ontogeny and in the postnatal thymus. This distinct expression is suggestive of functional importance for TEC biology. Genetic ablation of miR-205 in TECs, however, neither revealed a role for miR-205 in TEC function during homeostatic conditions nor during recovery from thymic stress conditions. Thus, despite its distinct expression, miR-205 on its own is largely dispensable for mTEC biology.     

Identification of Host-Targeted Small Molecules That Restrict Intracellular Mycobacterium tuberculosis Growth

Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.     

LIVER SINUSOIDAL CELLS : Identification of a Subpopulation for Erythrocyte Catabolism

Sequestration and degradation of red blood cells (RBC) are believed to occur in part in the liver, but the magnitude and cellular localization of this process remain uncertain. This problem was studied in rats by investigating isolated parenchymal and sinusoidal cell populations of the liver. After digesting the perfused liver with pronase, hepatic sinusoidal cells were isolated free of RBC and debris. Of the isolated cells, 90% were phagocytic, as judged by their uptake of colloidal ¹⁹⁸Au or of aggregated albumin-¹³¹I administered in vivo After administration of spherocytic (heat-treated) RBC, however, only about one quarter of the isolated cells were found to contain phagocytized RBC. This apparently distinct population of RBC-phagocytizing cells is designated as "erythrophagocytic (EP)" cells. The EP cell population was further characterized functionally by its specific phagocytosis of colloidal carbon and of ^99mtechnetium-sulfur colloid and histochemically by its peroxidase activity. The role of the EP population in the catabolism of RBC-hemoglobin was studied in isolated hepatic sinusoidal cells by assay of microsomal heme oxygenase (MHO), which is the inducible enzyme system that converts heme to bilirubin. The MHO activity of individual sinusoidal isolates was related directly to their content of EP cells Assay of the MHO activity of the whole spleen and of the total EP cell population of the liver suggested that these two tissues may be of comparable importance in their ability to degrade RBC-hemoglobin.     

Identification of a LIF-Responsive,Replication-Competent Subpopulation of Human β Cells

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Human Bone Marrow Is Comprised of Adipocytes with Specific Lipid Metabolism

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Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

Identification of a Novel Subpopulation of Tumor-Initiating Cells from Gemcitabine-Resistant Pancreatic Ductal Adenocarcinoma Patients

Pancreatic ductal adenocarcinoma is highly resistant to systemic chemotherapy. Although there are many reports using pancreatic cancer cells derived from patients who did not receive chemotherapy, characteristics of pancreatic cancer cells from chemotherapy-resistant patients remain unclear. In this study, we set out to establish a cancer cell line in disseminated cancer cells derived from gemcitabine-resistant pancreatic ductal adenocarcinoma patients. By use of in vitro co-culture system with stromal cells, we established a novel pancreatic tumor-initiating cell line. The cell line required its direct interaction with stromal cells for its in vitro clonogenic growth and passaging. Their direct interaction induced basal lamina-like extracellular matrix formation that maintained colony formation. The cell line expressed CD133 protein, which expression level changed autonomously and by culture conditions. These results demonstrated that there were novel pancreatic tumor-initiating cells that required direct interactions with stromal cells for their in vitro cultivation in gemcitabine-resistant pancreatic ductal adenocarcinoma. This cell line would help to develop novel therapies that enhance effects of gemcitabine or novel anti-cancer drugs.     

人白血病细胞系KG-1a中肿瘤干细胞样亚群细胞的初步研究

探讨人白血病细胞系KG-1a中是否存在具有肿瘤干细胞样生物学特性的亚群细胞.瑞氏染色和吖啶橙染色分别观察白血病细胞系KG-1a细胞形态和RNA含量:流式细胞术检测细胞周期分布;免疫组化和流式细胞术检测KG-1a细胞CD34的表达;流式细胞术检测CD34 CD38-亚群细胞;烟酸己可碱Hoechst33342染色后用荧光显微镜观察KG-1a细胞中侧群(side population,SP)样细胞所占比例.结果显示,白血病细胞系KG-1a细胞核仁易见,形态原始;部分细胞RNA含量低,细胞处于G0/G1期的比例占15.5%.绝大多数细胞的胞浆和胞膜皆表达CD34抗原,KG-1a中CD34 细胞占96.3%,CD34 CD38-亚群细胞占7.02%;SP样细胞大致比例为7.60%.本研究表明.人白血病细胞系KG-1a中存在具有肿瘤干细胞样生物学特性的亚群细胞.     

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.     

Identification of Tumor-Associated Antigens from Medullary Breast Carcinoma by a Modified SEREX Approach

Medullary breast carcinoma (MBC) is a relatively rare malignancy with heavy lymphocytic infiltration that despite cytologically anaplastic features and high mitotic index has more favorable prognosis than other types of breast cancer. Lymphocytic infiltration of tumors reflects ongoing immune response against tumor antigens which could represent a great interest as potential targets for cancer immunotherapy. The search for MBC antigens by SEREX methodology has not been successful due to a very high titer of false positive clones, representing immunoglobulin genes. Here, we describe a novel approach for generating cDNA expression libraries from MBC tumor samples which are depleted of IgG cDNA clones and, therefore, are suitable for the identification of novel tumor-associated antigens (TAA) by SEREX approach. Modified methodology allowed us to isolate a panel of known and novel TAA which are currently under further investigation.     

骨髓间充质干细胞成肌和成脂分化的调控

骨髓间充质干细胞(mesenchymal stem cells,MSCs)是来源于骨髓基质的一类具有高度自我更新能力和多向分化潜能的成体干细胞.因其具有容易获取、体外扩增方便迅速、移植排斥反应较弱等优点而成为临床应用的理想细胞模型.骨髓间充质干细胞向成肌和成脂的分化对动物机体内肌肉和脂肪的组成具有直接影响,因而与肉品质及人类健康息息相关.本文综述了骨髓间充质干细胞定向分化为骨骼肌细胞和脂肪细胞的过程及其调控机制,并重点分析了关键调控因子PRDM16(PR domain-containing16)和骨形态发生蛋白(bone morphogenetic proteins,BMPs)在骨髓间充质干细胞成肌和成脂分化中的作用.     

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine (4-amino-4,6-dideoxy-D -manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L -glycero-tetronyl)-2-O-methyl-D -perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).     

Identification of a Gene That Shortens Clonal Life Span of Paramecium Tetraurelia

We have isolated a Paramecium tetraurelia mutant that divides slowly in daily reisolation cultures and repeats short clonal life spans after successive autogamies. Here we show, using breeding analysis, that a recessive mutation is responsible for the low fission rate and that this low rate is closely related to the short clonal life span. We conclude that a single pleiotropic gene controls these traits and have named it jumyo. In an attempt to further characterize the jumyo mutant, we have revealed that it has a culture life span similar to that of the wild-type cells and that, when mass cultured, it can divide as rapidly as wild-type cells. There was strong evidence that the mutant cells excreted into culture medium some substance that promotes their cell division. These findings may not only present supporting evidence for the hypothesis that the cellular life span is genetically programmed but also give a material basis for the study of the controlling mechanism of cell division in relation to the clonal life span.     

Identification of a Protein That Inactivates the Competence-Stimulating Peptide of Streptococcus pneumoniae

Competence for genetic transformation of Streptococcus pneumoniae is a transient physiological property inducible by a competence-stimulating peptide (CSP). A 68-kDa CSP-inactivating protein was previously obtained following lithium chloride (LiCl) extraction. By the same protocol, a CSP-inactivating protein was purified and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as an endopeptidase, PepO. Analysis of a pepO mutant provided no support for the hypothesis that PepO participates in competence regulation. To reconcile in vitro and in vivo data, we suggest that LiCl treatment results in the release of intracellular molecules, including PepO.     

Proteome and Phosphoproteome Analysis of Brown Adipocytes Reveals That RICTOR Loss Dampens Global Insulin/AKT Signaling

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Highlights

• •Global and targeted phosphoproteomics in RICTOR-deficient brown adipocytes.
• •RICTOR loss leads to higher levels of many interferon response-associated proteins.
• •RICTOR loss dampens the dynamic insulin-dependent phosphoproteome response.
• •ACLY S455, VIM S39, and EIF4B S422 are among the most dampened phosphosites.