HTLV-I,infective dermatitis,and tropical spastic paraparesis

Since human T-cell lymphotropic virus (HTLV-I) was identified in 1980 as causing human disease, it has been etiologically associated with adult T-cell lymphoma/leukemia (ATL) and tropical spastic paraparesis (TSP). More recently, several new diseases have been reported in association with this virus, including infective dermatitis of Jamaican children, which we reported in 1990. Studies on infective dermatitis have shown that these children have abnormalities of immune function, and some develop other HTLV-I associated disorders, including TSP. This paper reviews the work done on infective dermatitis to date, and explores the association with TSP.     

RAPID SYNCYTIUM FORMATION BETWEEN HUMAN T-CELL LEUKAEMIA VIRUS TYPE-I (HTLV-I)-INFECTED T-CELLS AND HUMAN NERVOUS SYSTEM CELLS: A POSSIBLE IMPLICATION FOR TROPICAL SPASTIC PARAPARESIS/HTLV-I ASSOCIATED MYELOPATHY

Tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM), is characterized by infiltration of human T cell leukaemia virus type-I (HTLV-I)-infected T-cells, anti-HTLV-I cytotoxic T cells and macrophages into the patients’ cerebrospinal fluid and by intrathecally formed anti-HTLV-I antibodies. This implies that the disease involves a breakdown of the blood—brain barrier. Since astrocytes play a central role in establishing this barrier, the authors investigated the hypothesis that the HTLV-I infected T cells disrupt this barrier by damaging the astrocytes. The present study revealed the HTLV-I-producing T cells conferred a severe cytopatic effect upon monolayers of astrocytoma cell line in co-cultures. Following co-cultivation, HTLV-I DNA and proteins appeared in the monolayer cells, but after reaching a peak their level gradually declined. This appearance of the viral components was proved to result from a fusion of the astrocytic cells with the virus-producing T cells, whereas their subsequent decline reflected the destruction of the resulting syncytia. This fusion could be specifically blocked by anti HTLV-I Env antibodies, indicating that it was mediated by the viral Env proteins expressed on the surface of the virus-producing cells. Similar fusion was observed between the HTLV-I-producing cells and certain other human nervous system cell lines. If such fusion of HTLV-I-infected T cells occurs also with astrocytes and other nervous system cells in TSP/HAM patients, it may account, at least partially, for the blood—brain barrier breakdown and some of the neural lesions in this syndrome.     

Suppression of lymphocyte spontaneous proliferative response by proteolipid protein peptide in patients with HAM/TSP

To understand the immune mechanism suggested in HTLV-I-associated myelopathy (HAM/TSP), we investigated T cell response to proteolipid protein (PLP). Because of high autologous proliferative response (APR) of peripheral blood mononuclear cells (PBMC) in culture, the lymphocyte proliferation assay was not useful in this disease. Unexpectedly, however, APR was profoundly (70–98%) suppressed in 6 of 9 cases when PLP peptide 105-124 was added in the culture. PLP peptide 85-104 or 145-159 also suppressed APR in a few cases. Time course study showed that the peptide-mediated suppression became apparent after day 4 in culture. The results can be interpreted as that suppressor cells recognizing the PLP peptides were present in the PBMC of HAM/TSP patients and suppressed the APR as the consequence of antigen specific response. This may indicate that a T cell response to certain PLP determinants is involved in the pathomechanism of HAM/TSP at least in part. Molecular mimicry between PLP and HTLV-I mayaccount for the T cell sensitization to PLP in HAM/TSP.Special issue dedicated to Dr. Marjorie B. Lees.     

Genetic variability in the extracellular matrix protein as a determinant of risk for developing HTLV-I-associated neurological disease

Aggrecan, which is a well-known proteoglycan in joint cartilage, also exists in the spinal cord and plays an important role in maintaining water content in the extracellular matrix structure. In this study, we first examined the variable number of tandem repeat (VNTR) polymorphism of the aggrecan gene in 227 HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, in 217 HTLV-I-infected healthy carriers (HCs), and in 85 normal controls. The VNTR allele 28 (1,630 bp) was more frequently observed in HAM/TSP patients than in HCs (χ ²=12.02, p=0.0005, odds ratio 1.79, 95% C.I. 1.29–2.50) and in controls (χ ²=13.43, p=0.0002, odds ratio 2.54, 95% C.I. 1.52–4.25), although this allele was not related to disease progression or to HTLV-I provirus load. We also found that the aggrecan concentration in cerebrospinal fluid (CSF) from rapidly progressive HAM/TSP patients was significantly higher than in slowly progressive patients (corrected p=0.0145) but not in infected non-inflammatory neurological other disease controls (OND) (corrected p=0.078). We then analyzed this aggrecan VNTR polymorphism in the different set of patients with HAM/TSP (n=58) and healthy carriers (n=70). This analysis, again, revealed that allele 28 was detected more frequently in HAM/TSP group than in HCs (χ ²=11.03, p=0.0009, odd ratio 3.04, 95% C.I. 1.55–5.97). The reproducibility of our study was regarded as a second- or third-class association by comparing combined p values and the Better Associations for Disease and GEnes (BADGE) system. Our results suggest that aggrecan polymorphism can be a novel genetic risk factor for developing HAM/TSP.Financial support: Grant in Aid for Research on Brain Science of the Ministry of Health, Labor and Welfare, Japan.     

Design and synthesis of several small-size HTLV-I protease inhibitors with different hydrophilicity profiles

The human T cell leukemia/lymphotropic virus type 1 (HTLV-I) is clinically associated with adult T cell leukemia/lymphoma, HTLV-I associated myelopathy/tropical spastic paraparesis, and a number of other chronic inflammatory diseases. To stop the replication of the virus, we developed highly potent tetrapeptidic HTLV-I protease inhibitors. In a recent X-ray crystallography study, several of our inhibitors could not form co-crystal complexes with the protease due to their high hydrophobicity. In the current study, we designed, synthesized and evaluated the HTLV-I protease inhibition potency of compounds with hydrophilic end-capping moieties with the aim of improving pharmaceutic and pharmacokinetic properties.     

Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles

The human mucin MUC1 is expressed both as a transmembrane heterodimeric protein complex that recycles via the trans-Golgi network (TGN) and as a secreted isoform. To determine whether differences in cellular trafficking might influence the O-glycosylation profiles on these isoforms, we developed a model system consisting of membrane-bound and secretory-recombinant glycosylation probes. Secretory MUC1-S contains only a truncated repeat domain, whereas in MUC1-M constructs this domain is attached to the native transmembrane and cytoplasmic domains of MUC1 either directly (M0) or via an intermitting nonfunctional (M1) or functional sperm protein-enterokinase-agrin (SEA) module (M2); the SEA module contains a putative proteolytic cleavage site and is associated with proteins receiving extensive O-glycosylation. We showed that MUC1-M2 simulates endogenous MUC1 by recycling from the cell surface of Chinese hamster ovary (CHO) mutant ldlD14 cells through intracellular compartments where its glycosylation continues. The profiles of O-linked glycans on MUC1-S secreted by epithelial EBNA-293 and MCF-7 breast cancer cells revealed patterns dominated by core 2-based oligosaccharides. In contrast, the respective membrane-shed probes expressed in the same cells showed a complete shift to patterns dominated by sialyl core 1. In conclusion, glycan core profiles reflected the subcellular trafficking pathways of the secretory or membranous probes and the modifying activities of the resident glycosyltransferases.     

Damaged DNA and miscounted chromosomes: human T cell leukemia virus type I tax oncoprotein and genetic lesions in transformed cells

Genetic instability is a recurring theme in human cancers. Although the molecular mechanisms mediating this effect commonly observed in transformed cells are not completely understood, it has been proposed to involve either the loss of DNA repair capabilities or the loss of chromosomal stability. The transforming retrovirus human T cell leukemia virus type I (HTLV-I) encodes a viral oncoprotein Tax, which is believed to cause the genomic instability characteristic of HTLV-I-infected cells. This review focuses on the ability of HTLV-I Tax to disrupt the cellular processes of DNA repair and chromosomal segregation. The consequences of these effects as well as the evolutionary advantage this may provide to HTLV-I are discussed.     

Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, > 75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.     

Biochemical and morphological changes in the nuclear matrix prepared from apoptotic HL‐60 cells: Effect of different stabilizing procedures

Apoptotic cell death is characterized by deep morphological changes that take place in the nucleus. It is unclear whether modifications also occur in the nuclear matrix, a mainly proteinaceous structure that conceivably acts as a nuclear framework. We have investigated whether biochemical and morphological alterations of the nuclear matrix prepared from apoptotic HL‐60 cells were dependent on the manipulations to which isolated nuclei were subjected before DNase I digestion and 2 M NaCl extraction. Our results showed that the stabilizing procedures employed to preserve the inner fibrogranular network and nucleolar remnants of the matrix (i.e., a 37°C incubation; exposure to sodium tetrathionate at 4°C; exposure to sodium tetrathionate at 37°C) had no effect on the protein recovery of apoptotic nuclear matrices, which was always approximately two‐ to fivefold less than in control matrices. Moreover, one‐ and two‐dimensional gel analysis of nuclear matrix proteins showed that, in apoptotic samples, striking quantitative changes were present, as compared with controls. Once again, these changes were seen irrespective of the stabilizing procedures employed. Also, transmission electron microscope analysis showed similar morphological alterations in all types of apoptotic nuclear matrices. By contrast, the immunofluorescent distribution of the 240‐kDa NuMA protein seen in apoptotic samples was more sensitive to the stabilizing treatments. Our results indicate that the biochemical and morphological changes of the apoptotic nuclear matrix are largely independent of the isolation protocols and strengthen the contention that destruction of the nuclear matrix network is one of the key events leading to apoptotic nuclear destruction. J. Cell. Biochem. 74:99–110, 1999. © 1999 Wiley‐Liss, Inc.     

The vacuole system in stomatal cells ofAllium. vacuole movements and changes in morphology in differentiating cells as revealed by epifluorescence,video and electron microscopy

Summary The development of autofluorescent vacuoles in the stomatal cells ofAllium cepa andA. vineale was investigated using fluorescence microscopy of live cells, low light level television, cytochemistry and electron microscopy. During cell differentiation, the vacuole undergoes two major changes in morphology. In an intermediate form, it consists of a reticulum or network of interlinked tubules and small chambers. The network is formed from globular cisternae in very young GMCs and is maintained as a reticulum until it is transformed back into a globular form later in the differentiation of guard cells. The network thus remains intact through the course of one cell division. During its existence, the reticulum undergoes complex movements and rearrangements. The significance of these changes in the vacuole is discussed in terms of vacuole ontogeny and function and the mechanisms that control motility in plant cells.Abbreviations used DIC differential interference contrast - TV television - L³TV low light level television - GMC guard mother cell - I²V double image intensifier vidicon - I³V triple image intensifier vidicon - CCU camera control unit - SEG special effects generator - VTR video tape recorder - VCR video cassette recorder - TDG time-date generator - TBC time base corrector - SYNC synchronization - BL blanking - VD vertical drive - HD horizontal drive - PMT photomultiplier tube - ALC automatic light compensation - LED light emitting diode.     

Cell density-dependent decrease in cytoskeletal actin and myosin in cultured osteoblastic cells: correlation with cyclic AMP changes.

During bone development, osteoblasts form a contiguous layer along recently deposited osteoid and their morphology changes from fibroblast-like to cuboidal. In culture, similar changes occur with increased cell density. We examined the possible role of cyclic AMP in this process since cyclic AMP was reported to increase in fibroblasts with increased cell density and similar shape changes were seen in response to parathyroid hormone, which also increases cellular cyclic AMP in osteoblastic cells. Osteoblast-enriched rat calvaria cells were seeded at increasing density. The distribution between Triton X-100 extractable and nonextractable actin and myosin was estimated by polyacrylamide gel electrophoresis. Intracellular cyclic AMP was estimated by prelabeling the cellular ATP pool with 3H-adenine, followed by extraction and separation of 3H-cAMP by high-performance liquid chromatography. We found that osteoblastic cells contain about 40 pg actin and 5.3 pg myosin per cell. Around 60% of the actin and 70% of the myosin were in the nonextractable (crosslinked) form at cell densities of 10,000 to 50,000 cells per cm2. Above 50,000 cells/cm2, there was a cell density-dependent reduction in crosslinked actin and myosin and a concomitant increase in cellular cyclic AMP. A comparable rise in cyclic AMP, produced by incubation with phosphodiesterase inhibitors, and treatment with other agents that increase cyclic AMP produced a similar decrease in the level of cytoskeletal actin and myosin. Cytochalasin B treatment, through its effect on actin polymerization, produced similar changes in cell shape and cytoskeletal actin. The findings suggest that an elevation in intracellular cyclic AMP may play a role in the density-dependent changes in cell shape and microfilament organization observed in osteoblasts.     

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.     

哮喘患者外周血Treg和Th1/Th2的变化及其与哮喘病情的关系

目的：探讨哮喘患者外周血调节性T细胞（Treg）以及辅助性T细胞（Th1/Th2）的比例的变化,探讨其在哮喘的临床治疗中的作用。方法：80例哮喘患者（哮喘组）按临床表现分为急性发作期组（54例）和缓解期组（26例）,同时选择50例健康体检者。应用流式细胞仪检测上述各组外周血CD4＋CD25＋Foxp3＋Treg、CD4＋IFN-γ＋Th1和CD4＋IL-4＋Th2细胞水平,并进行统计学分析。结果：哮喘组CD4＋CD25＋Foxp3＋Treg水平亦明显低于正常对照组（P〈0.05。其中急性发作期组Treg水平明显低于缓解期组和正常对照组（P〈0.05）。而哮喘组Th1/Th2比值显著低于对照组（P〈0.05）,且在哮喘急性发作组中Th1/Th2比值显著低于缓解期组和正常对照组（P〈0.05）。结论：提示Treg和Th在哮喘的发生和发展中起着重要的作用。     

Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks

We have fused the signal anchor sequences of a rat sialyl transferase and a human galactosyl transferase along with the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) to the jellyfish green fluorescent protein (GFP) and transiently expressed the chimeric genes in tobacco leaves. All constructs targeted the Golgi apparatus and co-expression with DsRed fusions along with immunolabelling of stably transformed BY2 cells indicated that the fusion proteins located all Golgi stacks. Exposure of tissue to brefeldin A (BFA) resulted in the reversible redistribution of ST-GFP into the endoplasmic reticulum. This effect occurred in the presence of a protein synthesis inhibitor and also in the absence of microtubules or actin filaments. Likewise, reformation of Golgi stacks on removal of BFA was not dependent on either protein synthesis or the cytoskeleton. These data suggest that ER to Golgi transport in the cell types observed does not require cytoskeletal-based mechanochemical motor systems. However, expression of an inhibitory mutant of Arabidopsis Rab 1b (AtRab1b(N121I) significantly slowed down the recovery of Golgi fluorescence in BFA treated cells indicating a role for Rab1 in regulating ER to Golgi anterograde transport.     

TPA-induced differentiation and adhesion of U937 cells: changes in ultrastructure, cytoskeletal organization and expression of cell surface antigens

Incubation of the human promonocytic cell line U937 with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h resulted in differentiation into immature macrophage-like cells and was accompanied by marked morphological and functional changes. U937 cells which normally grow in suspension and show a smooth surface, extended pseudopodia and became adherent to each other and to the surface of the culture vessel. Concomitant with the TPA-induced adherence U937 cells ceased to proliferate. Our results show that phorbol ester-treated U937 cells exhibited markedly increased levels of fibronectin and of the cytoskeletal proteins actin, myosin and vimentin including a reorganization of actin and vimentin filaments. The induction of both cellular adherence and growth inhibition were accompanied by a significantly reduced level of cells expressing transferrin receptors and changes in cell surface antigen expression. Here, the expression of the leukocytefunction antigens (LFA-1), including CD11 and CD18 was markedly enhanced during phorbol ester-induced differentiation. TPA-treatment, however, failed to enhance the small amount of U937 cells expressing the monocyte/macrophage-specific CD14 antigen or expressing MHC class-II antigens. A detailed analysis of the CD14 cluster by 7 differential antibodies resulted in an induction of TM1, UCHM1, MEM15, My4, and 3C10, whereas the epitopes recognized by TM2 and Mo2 remained unaltered. Neither indomethacin nor interferon-gamma were capable of inducing a marked expression of these antigen epitopes in TPA-treated cells. Although these data demonstrate that during phorbol ester-induced differentiation U937 cells acquire many properties typically associated with macrophages, the failure to express marked levels of macrophage-specific cell surface antigens suggests a transition of U937 cells from a promonocytic to an immature macrophage intermediate state rather than into mature macrophage-like cells.     

Cytokine profile and cytoskeletal changes after herpes simplex virus type 1 infection in human trabecular meshwork cells

Uveitis caused by herpes simplex virus (HSV)-1 is characterized by increased intraocular pressure (IOP) in the presence of anterior chamber inflammation. Despite their clinical significance, the pathogenic changes associated with HSV-1 infection in trabecular meshwork (TM) cells, the key cell type regulating IOP, have not been completely elucidated. In this study, cytokine array analyses showed a significant stepwise increase in monocyte chemoattractant protein (MCP)-1 expression upon HSV-1 infection in TM cells (p < 0.05). HSV-1 infection led to downregulation of fibrogenic molecules (fibronectin, α-smooth muscle actin, connective tissue growth factor and TGF-β1). Notably, HSV-1 infection caused a significant increase in actin stress fibres, with a twofold increase in active RhoA, which was enhanced by treatment with TGF-β1 and inhibited by treatment with the Rho-kinase inhibitor, Y-27632. TM cells treated with MCP-1 exhibited a dose-dependent increase in actin stress fibres compared to untreated TM cells. Our study suggests that HSV-1 infection in TM cells increases cell contractile activity rather than fibrotic changes in the extracellular matrix (ECM) components. Taken together, these observations demonstrate the enhanced expression of MCP-1 and TM cell contractile activity upon HSV-1 infection and events with potential implications for the pathobiology of abrupt IOP elevation in HSV-1 anterior uveitis.     

支气管哮喘患者呼吸道菌群及FeNO、Th/Treg细胞水平变化

探究支气管哮喘患者呼吸道菌群及呼出气一氧化氮(FeNO)、Th17细胞与调节性T细胞(Treg)水平变化。

选取2017年5月至2019年8月我院呼吸内科收治的60例支气管哮喘患者为观察组, 选择同期35例健康者作为对照组。观察两组对象呼吸道菌群, FeNO、Th17细胞、Treg细胞, T细胞亚群及免疫球蛋白水平。

观察组患者共分离出87株细菌, 其中奈瑟菌检出率最高, 其次为肺炎链球菌、甲型链球菌、表皮葡萄球菌、韦荣球菌; 对照组对象共分离出35株细菌, 其中甲型链球菌检出率最高, 其次为表皮葡萄球菌、奈瑟菌、韦荣球菌、消化球菌。观察组患者甲型链球菌占比低于对照组(χ²=4.554, P=0.032), 其余菌群比较差异无统计学意义(均P > 0.05)。观察组患者FeNO、Th17细胞、Treg细胞水平及Th17/Treg均高于对照组, 差异有统计学意义(均P < 0.05)。观察组患者CD4⁺细胞、CD4⁺/CD8⁺低于对照组, 差异有统计学意义(均P < 0.05);而两组对象CD3⁺、CD8⁺细胞水平比较差异无统计学意义(均P > 0.05)。观察组患者免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)水平与对照组比较差异无统计学意义(均P > 0.05)。

支气管哮喘患者呼吸道检出率较高的菌群为奈瑟菌, 且FeNO、Th17/Treg水平均较高, 细胞免疫功能低下, 应及时进行治疗。

     

Renal cortical cells in primary monolayer culture. Enzymatic changes and morphological observations.

Stress fibers and bands of intermediate filaments (100 Å) were studied in cultured non-muscle cells using laser microbeam techniques. Wavelengths of 532, 537 and 280 nm were used, and no artificial chromophores were employed. Lesions were assayed using a combination of phase contrast, polarizing and transmission electron microscopy (TEM). (1) Stress fibers 1–2 μm in diameter were narrowed or completely servered by irradiation at 532, 537 and 280 nm. Stress fibers could be grouped into two classes: (a) those whose severed ends separated during the first few seconds following laser irradiation (46% of fibers irradiated); (b) those fibers which showed no movements (54%). Microtubules which paralleled stress fibers persisted in the presence of colcemid for up to 5 h, and alignment of the severed stress fiber ends was maintained even in their absence. Injured stress fibers appear to be repaired within 1 h of irradiation. (2) Bands of 100 Å filaments were induced in non-muscle cells in secondary cultures of neonatal rat heart by exposure to colcemid. Lesions which appeared as phase dense spots were induced in these bands by irradiation at 532, 537 and 280 nm. The positions of the lesions in the band relative to one another did not change over several hours despite movements of the entire band. These studies demonstrate that (a) stress fibers may be an excellent system in which to study subcellular repair; (b) induced bands of 100 Å filaments probably move passively in the cells containing them; (c) laser irradiation of cytoplasmic filaments in non-muscle cells does not require the introduction of an artificial chromophore.     

Profiling Eph receptor expression in cells and tissues: A targeted mass spectrometry approach

The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.