scholarly journals Exo-enzymatic addition of diazirine-modified sialic acid to cell surfaces enables photocrosslinking of glycoproteins

2021 ◽  
Author(s):  
Nageswari Yarravarapu ◽  
Rohit Sai Reddy Konada ◽  
Narek Darabedian ◽  
Nichole J. Pedowtiz ◽  
Soumya N. Krishnamurthy ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Cell Types ◽  
Cell Surfaces ◽  
Binding Interactions ◽  
Multiple Cell ◽  
Labeling Method ◽  
Cell Surface Glycoconjugates

Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ however use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here we report an exo-enzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAz-ylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

Polydiacetylene liposomes with phenylboronic acid tags: a fluorescence turn-on sensor for sialic acid detection and cell-surface glycan imaging

Nanoscale ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 4570-4578 ◽  
Author(s):  
Dong-En Wang ◽  
Jiahang Yan ◽  
Jingjing Jiang ◽  
Xiang Liu ◽  
Chang Tian ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Sialic Acid ◽  
Cell Surface ◽  
Living Cell ◽  
Phenylboronic Acid ◽  
Cell Surfaces ◽  
Turn On ◽  
Fluorescence Turn On ◽  
Free Sialic Acid

A new polydiacetylene (PDA) liposome-based sensor system was developed as a fluorescence turn-on sensor for the detection of free sialic acid (SA) in aqueous solution and in situ imaging of glycans on living cell surfaces.

Mucin mRNA Expression in Normal and Vasomotor Inferior Turbinates

1997 ◽  
Vol 11 (4) ◽  
pp. 293-302 ◽  
Author(s):  
Michelle R. Aust ◽  
Cathy S. Madsen ◽  
Anita Jennings ◽  
Jan L. Kasperbauer ◽  
Sandra J. Gendler
Keyword(s):  
Respiratory Tract ◽  
Expression Patterns ◽  
Inferior Turbinate ◽  
Cell Types ◽  
Upper Respiratory Tract ◽  
Vasomotor Rhinitis ◽  
Disease States ◽  
Multiple Cell ◽  
Nasal Secretions

Mucins are the major glycoprotein component of respiratory tract secretions. Little is known about their expression in the upper respiratory tract. In order to define this expression, in situ hybridization was performed on 19 normal and 4 vasomotor rhinitis (VMR) inferior turbinates to identify mucin mRNA. MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7 were expressed in both the normal and VMR turbinates. MUC 4 and MUC5AC were the most highly expressed mucins. MUC1, MUC2, MUC4, and MUC5AC were expressed mainly by the epithelial border, whereas MUC5B and MUC7 were expressed by the submucosal glands. MUC1 and MUC4 exhibited a diffuse expression by multiple cell types along the mucosal border, whereas MUC2 and MUC5AC expression appeared to be limited to a subpopulation of epithelial cells, most likely goblet cells. Although MUC1, MUC4, and MUC5AC showed sporadic submucosal glandular expression, MUC5B and MUC7 appeared to be the predominant submucosal gland mucins in the inferior turbinates. MUC3 and MUC6 expression, which have been found primarily in the gastric mucosa, were not seen in any of the inferior turbinate samples examined. The only difference seen between normal and VMR turbinates was a slight decrease in MUC1 expression in the VMR group. The variety of mucins expressed and the diversity of their expression patterns may have significance in terms of the rheologic and particle clearance properties of nasal secretions. Understanding the expression patterns in normal turbinates will serve as the foundation for further study of these mucins in disease states.

scholarly journals Isolation of an adhesion-mediating protein from chick neural retina adherons.

1985 ◽  
Vol 101 (3) ◽  
pp. 1071-1077 ◽  
Author(s):  
D Schubert ◽  
M LaCorbiere
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Cultured Cells ◽  
Cell Types ◽  
Neural Retina ◽  
Cell Surface Receptor ◽  
Isolation And Characterization ◽  
Surface Receptor ◽  
Adhesive Interactions

Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.

scholarly journals Differential expression of multiple cell-surface heparan sulfate proteoglycans during embryonic tooth development.

1994 ◽  
Vol 42 (8) ◽  
pp. 1043-1054 ◽  
Author(s):  
X M Bai ◽  
B Van der Schueren ◽  
J J Cassiman ◽  
H Van den Berghe ◽  
G David
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Differential Expression ◽  
Tooth Development ◽  
Multiple Forms ◽  
Dental Tissues ◽  
Cell Matrix ◽  
Heparin Sulfate ◽  
Multiple Cell

Heparan sulfate accumulates on cell surfaces and at cell-matrix interfaces, and functionally modulates several of the effector molecules that support the interactions, growth, and differentiation of developing tissues. Using heparin sulfate-specific monoclonal antibodies MAb, we obtained evidence that extracts from rodent embryos contain multiple forms of cell surface-associated heparan sulfate proteoglycan (PG). Taking tooth development in the mouse embryo as a model to further investigate the relevance of this PG redundancy and using MAb against heparan sulfate, antibodies specific for syndecan (syndecan-1) and fibroglycan (syndecan-2) (two distinct members of a larger family of cell-surface heparan sulfate PGs), and specific cDNA probes for these two cell-surface PGs, we obtained in situ evidence for regulated and differential expression of multiple cell-surface heparan sulfate PGs. The unique, distinctive, and coordinated changes in the expressions of these PGs during morphogenesis and differentiation of dental tissues suggest that the various cell-surface PGs are not truly redundant but play important, specific, and potentially complementary roles during embryonic development.

scholarly journals The mt1 Melatonin Receptor and RORβ Receptor Are Co-localized in Specific TSH-immunoreactive Cells in the Pars Tuberalis of the Rat Pituitary

2002 ◽  
Vol 50 (12) ◽  
pp. 1647-1657 ◽  
Author(s):  
Paul Klosen ◽  
Christele Bienvenu ◽  
Olivier Demarteau ◽  
Hugues Dardente ◽  
Hilda Guerrero ◽  
...  
Keyword(s):  
Cell Types ◽  
Orphan Receptor ◽  
Seasonal Effects ◽  
Pars Tuberalis ◽  
Melatonin Receptor ◽  
Functional Studies ◽  
Rat Pituitary ◽  
Mt1 Melatonin Receptor

The pars tuberalis (PT) of the pituitary represents an important target site for the time-pacing pineal hormone melatonin because it expresses a large number of mt1 receptors. Functional studies suggest that the PT mediates the seasonal effects of melatonin on prolactin (PRL) secretion. The aim of this study was the characterization of the pheno-type of melatonin-responsive cells. Furthermore, we determined whether RORβ, a retinoid orphan receptor present in the PT, was co-expressed in the same cells. We combined nonradioactive in situ hybridization (ISH) with hapten-labeled riboprobes for detection of the receptors and immunocytochemistry (ICC) for detection of αGSU (α-glycoprotein subunit), βTSH, βFSH, βLH, GH, PRL, and ACTH. Expression of mt1 mRNA was found in small round cells, co-localized with αGSU and βTSH. However, not all βTSH-containing cells expressed mt1 mRNA. The distribution of mt1- and RORβ-positive cells appeared to overlap, although more cells were labeled for RORβ than for mt1. Gonadotrophs, as well as other pars distalis cell types, were never labeled for mt1 melatonin receptor. Therefore, this study identifies the “specific” cells of the PT as the mt1 melatonin receptor-expressing cells.

scholarly journals Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

2019 ◽  
Author(s):  
Ryoji Amamoto ◽  
Mauricio D. Garcia ◽  
Emma R. West ◽  
Jiho Choi ◽  
Sylvain W. Lapan ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Rna Profiling ◽  
Complementary Method ◽  
Dissociated Cells ◽  
Multiple Cell ◽  
Heterogeneous Tissue

ABSTRACTRecent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labelled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as the Drosophila midgut. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

scholarly journals Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

2020 ◽  
Author(s):  
Shahar Alon ◽  
Daniel R Goodwin ◽  
Anubhav Sinha ◽  
Asmamaw T Wassie ◽  
Fei Chen ◽  
...  
Keyword(s):  
Splice Variants ◽  
Metastatic Breast ◽  
Cell Types ◽  
Specific Cell ◽  
Long Read ◽  
Multiple Cell ◽  
Cancer Biopsy ◽  
Subcellular Resolution ◽  
Mouse Visual Cortex

Abstract:Methods for highly multiplexed RNA imaging are limited in spatial resolution, and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to mouse brain, yielding readout of thousands of genes, including splice variants and novel transcripts. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in neurons of the mouse hippocampus, revealing patterns across multiple cell types; layer-specific cell types across mouse visual cortex; and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus ExSeq enables highly multiplexed mapping of RNAs, from nanoscale to system scale.One Sentence SummaryIn situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution.

scholarly journals Characterization of MAX.3 antigen, a glycoprotein expressed on mature macrophages, dendritic cells and blood platelets: identity with CD84

2000 ◽  
Vol 346 (3) ◽  
pp. 729-736 ◽  
Author(s):  
Stefan W. KRAUSE ◽  
Michael REHLI ◽  
Sven HEINZ ◽  
Reinhard EBNER ◽  
Reinhard ANDREESEN
Keyword(s):  
Dendritic Cells ◽  
Blood Platelets ◽  
Cell Types ◽  
Variable Expression ◽  
Surface Molecules ◽  
Sds Page ◽  
Molecular Masses ◽  
Antigen A

MAX.3 is a monoclonal antibody that preferentially reacts with mature macrophages (MAC), monocyte-derived dendritic cells, megakaryocytes and platelets. In this study, we describe the characterization, purification and identification of the MAX.3 antigen. Immunoprecipitation and SDS/PAGE revealed different molecular masses of MAX.3 antigen in MAC (60-90 kDa) and platelets (58-64 kDa), whereas a similar size (45 kDa) was observed in both cell types after digestion with N-glycosidase F. Lectin affinity and sequential treatment with different glycosidases suggests complex type glycosylation of MAX.3 antigen in MAC and hybrid type glycosylation in platelets. Amino acid sequencing led to the identification of a corresponding cDNA clone and showed its identity to the sequence of the CD84 antigen, a member of the CD2 family of cell surface molecules. MAX.3/CD84 was further studied by immunohistochemistry and a variable expression was found on tissue MAC, confirming this antigen to be mainly a marker for MAC in situ.

Dual Electrochemiluminescence Signal System for In Situ and Simultaneous Evaluation of Multiple Cell-Surface Receptors

2017 ◽  
Vol 9 (3) ◽  
pp. 2074-2082 ◽  
Author(s):  
Bin Zhou ◽  
Youyi Qiu ◽  
Qingqing Wen ◽  
Mingyao Zhu ◽  
Peihui Yang
Keyword(s):  
Cell Surface ◽  
Cell Surface Receptors ◽  
Signal System ◽  
Surface Receptors ◽  
Simultaneous Evaluation ◽  
Multiple Cell

scholarly journals Distribution of H-2 microenvironments in the mouse thymus. Immunoelectron microscopic identification of I-A and H-2K bearing cells.

1980 ◽  
Vol 28 (10) ◽  
pp. 1089-1099 ◽  
Author(s):  
W Van Ewijk ◽  
R V Rouse ◽  
I L Weissman
Keyword(s):  
Cell Types ◽  
Ultrastructural Level ◽  
Perivascular Spaces ◽  
Mouse Thymus ◽  
Histocompatibility Complex ◽  
Positive Elements ◽  
Reticular Cells ◽  
Labeling Method ◽  
T Cell Maturation

Antigens coded for by the major histocompatibility complex (MHC) are differentially expressed in the mouse thymus. Immunoperoxidase studies of frozen thymus sections incubated with monoclonal (hybridoma) anti-I-Ak antibodies revealed a dendritic straining pattern in the cortex and a confluent staining pattern in the medulla. Serial sections incubated with monoclonal anti-H-2Kk antibodies showed that H-2Kk antigens were only present at detectable levels in the medulla. Microenvironments expressing H-2Kk antigens also expressed I-Ak antigens. In cortico-medullary regions, relatively large MHC-negative areas were found. These areas appeared to connect to perivascular spaces surrounding blood vessels. Using a new postfixation labeling method for the detection of cell surface associated antigens on cells of the lymphoid system in situ, we have characterized the nature of MHC positive cell types at the ultrastructural level. These studies show that epithelial-reticular cells are the major MHC positive elements in the thymus. Lymphocytes in the medulla and in cortico-medullary bounderies are also MHC positive, however, lymphocytes in the cortex were not detectably labeled. These findings support the contention that epithelial-reticular cells are involved in the H2-restriction process during T cell maturation.

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