Pattern Of Cell Surface Proteins Research Articles

A subunit of the oligosaccharyltransferase complex is required for interspecific gametophyte recognition in Arabidopsis.

Species-specific gamete recognition is a key premise to ensure reproductive success and the maintenance of species boundaries. During plant pollen tube (PT) reception, gametophyte interactions likely allow the species-specific recognition of signals from the PT (male gametophyte) by the embryo sac (female gametophyte), resulting in PT rupture, sperm release, and double fertilization. This process is impaired in interspecific crosses between Arabidopsis thaliana and related species, leading to PT overgrowth and a failure to deliver the sperm cells. Here we show that ARTUMES (ARU) specifically regulates the recognition of interspecific PTs in A. thaliana. ARU, identified in a genome-wide association study (GWAS), exclusively influences interspecific—but not intraspecific—gametophyte interactions. ARU encodes the OST3/6 subunit of the oligosaccharyltransferase complex conferring protein N-glycosylation. Our results suggest that glycosylation patterns of cell surface proteins may represent an important mechanism of gametophyte recognition and thus speciation.

Molecular Imaging of Pancreatic Cancer with Antibodies

Development of novel imaging probes for cancer diagnostics remains critical for early detection of disease, yet most imaging agents are hindered by suboptimal tumor accumulation. To overcome these limitations, researchers have adapted antibodies for imaging purposes. As cancerous malignancies express atypical patterns of cell surface proteins in comparison to noncancerous tissues, novel antibody-based imaging agents can be constructed to target individual cancer cells or surrounding vasculature. Using molecular imaging techniques, these agents may be utilized for detection of malignancies and monitoring of therapeutic response. Currently, there are several imaging modalities commonly employed for molecular imaging. These imaging modalities include positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance (MR) imaging, optical imaging (fluorescence and bioluminescence), and photoacoustic (PA) imaging. While antibody-based imaging agents may be employed for a broad range of diseases, this review focuses on the molecular imaging of pancreatic cancer, as there are limited resources for imaging and treatment of pancreatic malignancies. Additionally, pancreatic cancer remains the most lethal cancer with an overall 5-year survival rate of approximately 7%, despite significant advances in the imaging and treatment of many other cancers. In this review, we discuss recent advances in molecular imaging of pancreatic cancer using antibody-based imaging agents. This task is accomplished by summarizing the current progress in each type of molecular imaging modality described above. Also, several considerations for designing and synthesizing novel antibody-based imaging agents are discussed. Lastly, the future directions of antibody-based imaging agents are discussed, emphasizing the potential applications for personalized medicine.

Epigenetic regulators governing cancer stem cells and epithelial-mesenchymal transition in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is amongst the most prevalent form of cancer worldwide with its predominance in the Indian subcontinent due to its etiological behavioral pattern of tobacco consumption. Late diagnosis, low therapeutic response and aggressive metastasis are the foremost confounders accountable for the poor 5 year survival rate of OSCC. These failures are attributed to the existence of "Cancer Stem cell (CSC)" subpopulation within the tumour environment. Quiescence, apoptotic evasion, resistance to DNA damage, abnormal expression of drug transporter pumps and in vivo tumorigenesis are the defining hallmarks of CSC phenotype. These CSCs have been distinguished from the tumor mass by determining the expression patterns of cell surface proteins, specific stemness markers and quantifying the cellular activities such as drug efflux & aldehyde dehydrogenase activity. Hence, it is necessary to understand the underlying mechanisms that regulate the CSC features in tumor development, metastasis and response to chemotherapy. Increasing evidence suggests that majority of malignant cells eventually undergoing Epithelial-Mesenchymal transition (EMT) share many biological characteristics with CSCs. Thus, this review encompasses the functional relevance of CSC and EMT markers in OSCC population with a hope to elucidate the fundamental mechanisms underlying cancer progression and to highlight the most relevant epigenetic mechanisms that contribute to the regulation of CSC features. We further aimed to explore the causal effects of nicotine, a major tobacco carcinogen, on epigenetic mechanisms regulating the OSCC CSCs and EMT markers which unravels the undisputable contribution of tobacco in oral carcinogenesis.

Phenotypic, ultra-structural, and functional characterization of bovine peripheral blood dendritic cell subsets.

Dendritic cells (DC) are multi-functional cells that bridge the gap between innate and adaptive immune systems. In bovine, significant information is lacking on the precise identity and role of peripheral blood DC subsets. In this study, we identify and characterize bovine peripheral blood DC subsets directly ex vivo, without further in vitro manipulation. Multi-color flow cytometric analysis revealed that three DC subsets could be identified. Bovine plasmacytoid DC were phenotypically identified by a unique pattern of cell surface protein expression including CD4, exhibited an extensive endoplasmic reticulum and Golgi apparatus, efficiently internalized and degraded exogenous antigen, and were the only peripheral blood cells specialized in the production of type I IFN following activation with Toll-like receptor (TLR) agonists. Conventional DC were identified by expression of a different pattern of cell surface proteins including CD11c, MHC class II, and CD80, among others, the display of extensive dendritic protrusions on their plasma membrane, expression of very high levels of MHC class II and co-stimulatory molecules, efficient internalization and degradation of exogenous antigen, and ready production of detectable levels of TNF-alpha in response to TLR activation. Our investigations also revealed a third novel DC subset that may be a precursor of conventional DC that were MHC class II+ and CD11c−. These cells exhibited a smooth plasma membrane with a rounded nucleus, produced TNF-alpha in response to TLR-activation (albeit lower than CD11c+ DC), and were the least efficient in internalization/degradation of exogenous antigen. These studies define three bovine blood DC subsets with distinct phenotypic and functional characteristics which can be analyzed during immune responses to pathogens and vaccinations of cattle.

Comparison between fibroblasts and mesenchymal stem cells derived from dermal and adipose tissue

Stem cells have the ability to renew themselves and differentiate into various cell types. For this reason, numerous research groups have been studying these cells for their therapeutic potential. Some of the therapies, however, are not producing the expected results because of contamination by other cell types, especially by fibroblasts. In the cosmetic industry, stem cells are used to test the efficacy of anti-ageing and rejuvenation products. The purpose of this work was to gain a better understanding of the differences in phenotype, in gene expression associated with stem cells, in the pattern of cell surface proteins and in the differentiation capacity of adipose-derived stem cells, of skin-derived stem cells and of commercially available fibroblasts. In this study, we compared fibroblasts with mesenchymal stem cells derived from bone marrow, skin (dermis) and adipose tissue, to assess the differentiation potential of fibroblasts. Dermal and adipose stem cells were isolated from aesthetic surgery patients, and fibroblasts were obtained from a commercial source. The following parameters were used in this study: immunophenotypic profile (positive: CD29, CD73, CD90 and CD105; negative: CD14, CD45 and HLA-DR); differentiation into osteoblastic, chondrogenic and adipogenic cell types; and PCR array to analyse the gene expression of cells isolated from different culture passages. Fibroblasts express the same cell immunophenotypic markers, as well as the genes that are known to be expressed in stem cells, and were shown to be expressed also in adipose and dermis stem cells. Fibroblasts are also able to differentiate into the three cell lineages mentioned above, that is, adipocytes, osteocytes and chondrocytes. Human dermal fibroblasts have a potential to adhere to plastic surfaces and differentiate into other cell types. However, for stem cells intended to be used in cosmetics, experiments conducted with contaminated fibroblasts may produce poor or even falsely negative results for the efficacy of the active ingredient or formulation and thus conceal their promising effects as anti-ageing and skin rejuvenation products.

Heterobivalent ligands target cell-surface receptor combinations in vivo

A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and specificity. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantified by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.

CTCF and cohesin help neurons raise their self-awareness

In the brain neurons form extensively intermingled projections and synaptic interactions between them. To achieve proper neural connectivity, interactions between sections belonging to the same neuron are avoided. However, how can a cell tell, when it encounters another neuronal projection, whether it is part of the same cell? In other words, how do neurons become self-aware? Work of the last several years has revealed a complex system of self-recognition that involves expression of variable and cell-specific sets of cell-surface proteins (1). These sets constitute unique “barcodes” for each neuron and serve as molecular readouts of cell identity so that cells can distinguish between “self” and “non-self.” In PNAS, Monahan et al. (2) present data that provide insights into the molecular process by which otherwise identical cells can express unique “barcodes” of membrane proteins. Their results point to a role of large-scale chromatin architecture in setting and maintaining stochastic and cell-specific expression patterns of cell-surface proteins.

Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

BackgroundIt has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells.Methods and Principal FindingsSurface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed.Conclusions/SignificanceOur findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells.

More than Markers: Biological Significance of Cancer Stem Cell-Defining Molecules

Small populations within an increasing array of solid tumors, labeled cancer stem cells (CSC) or tumor-initiating cells (TIC), have the ability to differentiate, self-renew, and replicate the original tumor in vivo. To date, these cells have been distinguished from the bulk-tumor population by the expression pattern of cell-surface proteins (e.g., CD24, CD44, CD133) and cellular activities, such as the efflux of Hoechst dye or aldehyde dehydrogenase activity. Recent data have shown that these markers are inducible by exposure to anticancer agents; this finding highlights not only the potential fluidity of the CSC compartment, but also the functionality of these markers. The involvement of CD44 in invasion, adhesion, and metastasis, or the role of CD24 in modulation of src, FAK, and GLI1 are examples of these relevant roles. Instead of looking solely at the marker expression in these populations, we hope to clarify the biologically significant roles these markers and activities play in tumor progression, metastases, and as possible targets for therapy.

Tc17 CD8 T Cells: Functional Plasticity and Subset Diversity

IL-17-secreting CD8 T cells (Tc17) have been described in several settings, but little is known regarding their functional characteristics. While Tc1 cells produced IFN-gamma and efficiently killed targets, Tc17 cells lacked lytic function in vitro. Interestingly, the small numbers of IFN-gamma-positive or IL-17/IFN-gamma-double-positive cells generated under Tc17 conditions also lacked lytic activity and expressed a similar pattern of cell surface proteins to IL-17-producing cells. As is the case for Th17 (CD4) cells, STAT3 is important for Tc17 polarization, both in vitro and in vivo. Adoptive transfer of highly purified, Ag-specific IL-17-secreting Tc17 cells into Ag-bearing hosts resulted in near complete conversion to an IFN-gamma-secreting phenotype and substantial pulmonary pathology, demonstrating functional plasticity. Tc17 also accumulated to a greater extent than did Tc1 cells, suggesting that adoptive transfer of CD8 T cells cultured in Tc17 conditions may have therapeutic potential for diseases in which IFN-gamma-producing cells are desired.

Pediatric T-ALL but Not cALL Blasts Express Ligands for Siglec-7 Inhibiting NK Cell Mediated Lysis.

Changes in the glycosylation pattern of cell surface proteins occur during early stages of malignant transformation. These alterations in the glycan repertoire may contribute to numerous processes of tumor cell survival such as adhesion, migration and immune evasion. Sialic acid is the most common terminal carbohydrate moiety of glycan structures in humans binding to a family of eleven receptors termed sialic acid binding Ig-like lectins (siglecs). Siglecs are expressed on virtually all effector cells of the immune system and several members function as inhibitory receptors. Siglec-7 is known as an inhibitory receptor expressed by NK cells and T cells. Therefore, siglec-7 ligands may contribute to immune evasion of malignant cells.In order to analyze the expression of siglec-7 ligands by malignant cells, we cloned the extracellular domain of human siglec-7. The recombinant protein was expressed in 293 cells to allow glycosylation, which is supposed to be important for specificity. As the interaction of a single glycan and lectin is of low affinity, we tetramerized siglec-7 in analogy to MHC-tetramers to increase avidity. This strategy allowed for reliable use of siglec-7 fusion protein in flow cytometry. Analysis of PBMC from healthy donors revealed that siglec-7 ligands are expressed on all subpopulations of PBMC: expression was detected on CD4+ and CD8+ T-cells as well as on monocytes and NK cells. B cells could be subdivided into a positive and negative population after staining with siglec-7 tetramers.Subsequently, we analyzed primary lymphatic blasts from childhood leukemias. Interestingly, T-ALL blasts expressed ligands for siglec-7, whereas these ligands were absent from cALL blasts. As cALL blasts are known to be better targets for NK cell-mediated cytotoxicity than T-ALL blasts, we hypothesized that siglec-7 ligands on the surface of leukemic blasts inhibit NK cells. To test the functional relevance of siglec-7 ligand expression for NK cell-mediated cytotoxicity, we used soluble monomeric siglec-7 as competitive inhibitor in cytotoxicity assays. In these competition assays, NK cell-mediated specific lysis of leukemic cells increased roughly twofold in the presence of soluble siglec-7.These results show that engagement of inhibitory siglecs on the surface of effector cells might contribute to immune escape mechanisms of malignant cells. It underlines the function of siglec-7 as a non-MHC-specific inhibitory receptor on NK cells and the important role of altered glycans expression on malignant cells in tumor immunology.

Modulation of cell surface protein expression by infection with HIV-1.

The examples discussed above show the profound influence of HIV infection on expression pattern of cell surface proteins and the functional relevance thereof. Altered cell surface pattern is involved in all aspects of HIV-induced pathogenesis such as viral spreading viral adhesion and cellular apoptosis and is an important parameter for therapeutical approaches. The regulatory mechanism is not homogenous for all proteins but includes divergent effects like modulation of gene transcription and proteolytic cleavage. Modulation by viral infection might be either a direct or an indirect effect. Various viral proteins have been implicated in direct modulation like the regulatory proteins Tat, Nef and Vpu, but also the envelope proteins gp 120 and gp41. In addition, infection by HIV-1 has been shown to modulate expression of various cytokines including IL-10 and IFN-gamma. The altered expression of various surface proteins might be an indirect effect of cytokines acting on B cells, T cells and monocytic cells. By virus capture assays the presence of further proteins on viral surface was demonstrated indicating a possible function for viral life cycle. To study the modulation of expression of those additional important surface molecules by HIV and its biological function for the pathogenesis will be the aim of further studies in our laboratory.

Changes in the pattern of cell surface proteins during transformation of bloodstream forms of Trypanosoma brucei in vitro

Transformation of coated bloodstream forms of Trypunosomu hrircei t o procyclic forms in vitro occurs over a period of 4-5 days 111, during which time the parasite changes its morphology and adapts its metabolism to life within the insect mid-gut [2,3]. One of the best characterized events during transformation is the loss o f the variable surface glycoprotein (VSG) that constitutes the dense surface coat of bloodstream forms of the parasite [4]. However, very little is known about the membrane proteins that are located beneath the VSG; for example, it is not clear whether these other 'masked' antigens are also shed during transformation or whether they remain on the surface of the procyclic forms. In this present report, we have used lactoperoxidasecatalysed radioiodination of the cell surface to label proteins that are located underneath the VSG of bloodstream forms and to follow the change in composition of the surface antigen population at various stages during transit through the transformation pathway.

Changes in pattern and accessibility for 125I-labelling of cell-surface proteins after mesenchymal differentiation of embryonal carcinoma cells

Cell-surface proteins of the embryonal carcinoma line C17-S1 1003 (1003) and of some of its mesenchymal derivatives were studied. The surface proteins were labelled with 125I using the lactoperoxidase-glucose-glucose oxidase system either on the cells attached to the culture dishes or after their dissociation. Iodinated proteins were analyzed by two-dimensional gel electrophoresis. The patterns obtained with embryonal carcinoma cells 1003 and with two mesenchymal cell types derived from them, namely embryonic mesenchymal cells (line 10035) and fibroblastic cells (line 10031), were different one from the other, especially when considering the group of proteins labelled on the attached cells. The pattern of cell-surface proteins of the myoblastic line 1168, also derived from C17-S1, was found to be similar to that of 10031 fibroblastic cells. This result is discussed in the light of the phenotypic transition toward myogenesis, which can be obtained with 10031 fibroblastic cells but not with 10035 embryonic mesenchymal cells. A direct method of detection of lectin-binding proteins permitted us to identify the major concanavalin A-binding proteins. Two of them are common to all cell lines studied. They were labeled with 125I on the attached undifferentiated 1003 cells, while in all differentiated derivatives they became available for labelling after the cell detachment only.

Separation of mammalian cell surface proteins by a two-dimensional gel electrophoresis system

Separation of externally exposed plasma membrane proteins of mammalian cells has been achieved by a new two-dimensional gel electrophoresis system. The proteins were separated in the first dimension on cylindrical polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS) and in the second dimension on polyacrylamide slab gels containing 9 M urea, 0.1% SDS, and 0.1% Triton CF10. Using this method we have obtained reproducible high-resolution patterns of cell surface proteins of differentiated rat neuro-tumor cells in culture and of normal rat retinal cells. Different cell types show characteristic cell surface proteins in addition to ubiquitous ones. The number of common surface proteins between two cell types account for approximately half of the total surface proteins. By immunoprecipitation we have also found that rabbit anti-serum against a rat neuronal cell line can recognize most of these external proteins. Since the separation in the first dimension is done in the presence of SDS and the second dimension in the presence of SDS, a non-ionic detergent, and urea, the technique is particularly suitable for proteins that are of poor solubility. In addition to size, net charge and hydrophobicity appear to be important factors in the separation. Virtually all of the proteins that run in the first dimension can be recovered and further separated in the second.