Spatially marking and quantitatively counting membrane immunoglobulin M in live cells via Ag cluster–aptamer probes

2014 ◽  
Vol 50 (27) ◽  
pp. 3560 ◽  
Author(s):  
Ru Liu ◽  
Jiao Zhai ◽  
Li Liu ◽  
Yaling Wang ◽  
Yueteng Wei ◽  
...  
Keyword(s):  
Immunoglobulin M ◽  
Live Cells ◽  
Membrane Immunoglobulin ◽  
Ag Cluster

scholarly journals The membrane immunoglobulin receptor utilizes a Shc/Grb2/hSOS complex for activation of the mitogen-activated protein kinase cascade in a B-cell line

1995 ◽  
Vol 307 (1) ◽  
pp. 215-223 ◽  
Author(s):  
G Kumar ◽  
S Wang ◽  
S Gupta ◽  
A Nel
Keyword(s):  
Protein Kinase ◽  
B Cell ◽  
Map Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Adaptor Protein ◽  
Immunoglobulin M ◽  
Membrane Immunoglobulin ◽  
Mitogen Activated Protein ◽  
Kinase Cascade

Ligation of membrane immunoglobulin M (mIgM) receptor in the Ramos B-cell line induced tyrosine phosphorylation of several intracellular substrates, including the adaptor protein. Shc. Phosphorylated Shc could be seen to associate with Grb2 in a complex which included hSOS. Inasmuch as hSOS is involved in p21ras activation, we also demonstrated that mIgM ligation activated a Ras-dependent kinase cascade in which sequential activation of Raf-1 and MEK-1 culminates in the activation of p42 mitogen-activated protein (MAP) kinase (ERK-2). The tumour promoter and protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA), also activated Raf-1, MEK-1, and MAP kinase in Ramos cells, but did not induce tyrosine phosphorylation of Shc or Shc/Grb2 association. Okadaic acid, another tumour promoter and serine/threonine phosphatase inhibitor, activated p42 MAP kinase without activating Raf-1 or MEK-1, suggesting the existence of a serine/threonine phosphatase which directly regulates MAP kinase activity.

Ag cluster–aptamer hybrid: specifically marking the nucleus of live cells

2011 ◽  
Vol 47 (43) ◽  
pp. 11960 ◽  
Author(s):  
Zhipeng Sun ◽  
Yaling Wang ◽  
Yueteng Wei ◽  
Ru Liu ◽  
Huarui Zhu ◽  
...  
Keyword(s):  
Live Cells ◽  
Ag Cluster

scholarly journals Remyelination-Promoting DNA Aptamer Conjugate Myaptavin-3064 Binds to Adult Oligodendrocytes In Vitro

2020 ◽  
Vol 13 (11) ◽  
pp. 403
Author(s):  
Mahboubeh Fereidan-Esfahani ◽  
Wei Ying Yue ◽  
Brandon Wilbanks ◽  
Aaron J. Johnson ◽  
Arthur E. Warrington ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Immunoglobulin M ◽  
Dna Aptamer ◽  
Live Cells ◽  
Embryonic Mouse ◽  
Adult Rat ◽  
Cortical Cultures ◽  
Mixed Cortical Cultures

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.

scholarly journals Okadaic acid activates p42 mitogen-activated protein kinase (MAP kinase; ERK-2) in B-lymphocytes but inhibits rather than augments cellular proliferation: contrast with phorbol 12-myristate 13-acetate

1993 ◽  
Vol 290 (2) ◽  
pp. 545-550 ◽  
Author(s):  
A M Casillas ◽  
K Amaral ◽  
S Chegini-Farahani ◽  
A E Nel
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
B Lymphocytes ◽  
Okadaic Acid ◽  
Cellular Proliferation ◽  
Thymidine Incorporation ◽  
Immunoglobulin M ◽  
Kinase Activation ◽  
Membrane Immunoglobulin ◽  
Mitogen Activated Protein

Ligation of the membrane immunoglobulin M receptor as well as stimulation with the protein kinase C agonist phorbol 12-myristate 13-acetate leads to a B-lymphocyte proliferation and differentiation. Both stimuli activate p42 mitogen-activated protein (MAP) kinase in human B-lymphocytes [Casillas, Hanekom, Williams, Katz and Nel (1991) J. Biol. Chem. 266, 19088-19094]. MAP kinase activation is dependent on tyrosine as well as threonine phosphorylation of the kinase and its activity is inhibited by tyrosine as well as threonine/serine phosphatases. Okadaic acid, a specific inhibitor of type 1 and 2A serine/threonine phosphatases, induced MAP kinase activity in a potent and dose-dependent fashion, but failed to induce [3H]thymidine incorporation into normal human tonsil B-cells. Moreover, in combination with membrane immunoglobulin M ligation, okadaic acid decreased rather than increased [3H]thymidine incorporation. The kinetics of MAP kinase activation by okadaic acid differed from phorbol 12-myristate 13-acetate and anti-membrane immunoglobulin M stimulation. Okadaic acid induced tyrosine phosphorylation of 42 kDa and 44 kDa proteins which co-electrophoresed and co-chromatographed with ERK-2 and ERK-1 respectively. Ramos cells also contained a constitutively active 46 kDa MAP kinase which appeared as a separate peak in chromatography and could be immunoprecipitated by an antiserum against a rat ERK-1 fusion protein.

scholarly journals The structural basis for distinct binding avidity of Pertuzumab and Trastuzumab IgM towards HER2

2019 ◽  
Author(s):  
Firdaus Samsudin ◽  
Joshua Yi Yeo ◽  
Samuel Ken-En Gan ◽  
Peter J. Bond
Keyword(s):  
Dissociation Constants ◽  
Therapeutic Potential ◽  
Growth Factor Receptor ◽  
Multiscale Simulation ◽  
Immunoglobulin M ◽  
Live Cells ◽  
Structural Basis ◽  
Her2 Positive ◽  
Stable Mode ◽  
Future Design

SUMMARYHarnessing the therapeutic potential of immunoglobulin M (IgM) is of considerable interest in immunotherapy due to its complement-activating and cell-agglutinating abilities. Pertuzumab and Trastuzumab are monoclonal antibody drugs used in therapy for patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer but exhibit significantly different binding affinities as IgM when compared to the original IgG1 form. While the affinity of Pertuzumab IgM to the HER2 extracellular domain is about one order of magnitude higher than IgG1 in experiments, it was recently reported that Trastuzumab IgM and IgG have similar equilibrium dissociation constants to one another. We now perform an integrative multiscale simulation study in order to understand the structural basis for the differences in behavior between the two antibodies, based on complete antibody assemblies. We show that Pertuzumab IgM is able to utilize all of its V-regions to engage HER2 in a more stable mode than Trastuzumab IgM due to steric clashes between the large globular HER2 domains when bound to Trastuzumab. This is subsequently validated by confirming that Pertuzumab IgM inhibits proliferation in HER2 over-expressing live cells more effectively than its IgG1 counterpart. Given the widespread clinical use of Trastuzumab and Pertuzumab, elucidating the molecular details of antibody-antigen interaction may help guide the choice of epitopes for future design and selection of improved therapeutic antibody isotypes.

Electron Microscopic identification of Pre-B Lymphocytes in the Bone Marrow of a Patient with Chronic Myelocytic Leukemic in Blast Crisis

1982 ◽  
Vol 40 ◽  
pp. 346-347
Author(s):  
T. Mullin ◽  
G. Yee ◽  
M. Aheam ◽  
J. Trujillo
Keyword(s):  
Blast Crisis ◽  
Immunoglobulin M ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Transformation Theory ◽  
Electron Microscopic ◽  
Chemotherapeutic Sensitivity ◽  
Microscopic Studies ◽  
Hematopoietic Precursor ◽  
Lymphoblastic Transformation ◽  
B Lineage

There have been numerous reports in the current literature suggesting that hematopoietic precursor cells in some human chronic myelocytic leukemias (CML) undergo lymphoblastic transformation at the time of the acute blast crisis (BC) stage. The primary evidence offered in support of this transformation theory--lymphoblastic appearing morphology, increased terminal deoxynucleotidyl transferase (TdT) activity, and chemotherapeutic sensitivity to vincristine and prednisone--has been indirect, however, since these features may occur in nonlymphoid cells. More direct support for the Pre-B lineage of these cells has recently been provided by immunofluorescent light microscopic studies demonstrating the presence of intracytoplasmic immunoglobulin M (IgM) in these CML-BC cells.

4-dimensional, high-resolution imaging of living cells

1992 ◽  
Vol 50 (1) ◽  
pp. 416-417
Author(s):  
Shinya Inoué
Keyword(s):  
Light Microscope ◽  
Living Cells ◽  
Live Cells ◽  
Video Tape ◽  
Active Living ◽  
High Resolution Imaging ◽  
3 Dimensional ◽  
Dynamic Architecture ◽  
Resolution Imaging ◽  
Disk Memory

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

High-resolution scanning electron microscopy in biology

1990 ◽  
Vol 48 (3) ◽  
pp. 14-15
Author(s):  
Keiichi Tanaka
Keyword(s):  
High Resolution ◽  
Immunoglobulin M ◽  
Metal Coating ◽  
Biological Macromolecules ◽  
Ultrahigh Resolution ◽  
Preparation Methods ◽  
Low Contrast ◽  
Almost All ◽  
Charging Effect ◽  
Scanning Electron

With the development of scanning electron microscope (SEM) with ultrahigh resolution, SEM became to play an important role in not only cytology but also molecular biology. However, the preparation methods observing tiny specimens with such high resolution SEM are not yet established.Although SEM specimens are usually coated with metals for getting electrical conductivity, it is desirable to avoid the metal coating for high resolution SEM, because the coating seriously affects resolution at this level, unless special coating techniques are used. For avoiding charging effect without metal coating, we previously reported a method in which polished carbon plates were used as substrate. In the case almost all incident electrons penetrate through the specimens and do not accumulate in them, when the specimens are smaller than 10nm. By this technique some biological macromolecules including ribosomes, ferritin, immunoglobulin G were clearly observed.Unfortunately some other molecules such as apoferritin, thyroglobulin and immunoglobulin M were difficult to be observed only by the method, because they had very low contrast and were easily damaged by electron beam.

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