scholarly journals A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo.

1983 ◽  
Vol 97 (5) ◽  
pp. 1467-1475 ◽  
Author(s):  
J Wehland ◽  
M C Willingham ◽  
I V Sandoval
Keyword(s):  
Monoclonal Antibody ◽  
Biochemical Characterization ◽  
Mammalian Species ◽  
Antigenic Site ◽  
In Vivo Studies ◽  
Alpha Tubulin ◽  
3T3 Fibroblasts ◽  
Microtubule Polymerization

The antigenic site recognized by a rat monoclonal antibody (clone YL 1/2) reacting with alpha-tubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) has been determined and partially characterized. YL 1/2 reacts specifically with the tyrosylated form of brain alpha-tubulin from different mammalian species. YL 1/2 reacts with the synthetic peptide Gly-(Glu)3-Gly-(Glu)2-Tyr, corresponding to the carboxyterminal amino acid sequence of tyrosylated alpha-tubulin, but does not react with Gly-(Glu)3-Gly-(Glu)2, the constituent peptide of detyrosylated alpha-tubulin. Electron microscopy as well as direct and indirect immunofluorescence microscopy shows that YL 1/2 binds to the surface of microtubules polymerized in vitro and in vivo. Further in vitro studies show that the antibody has no effect on the rate and extent of microtubule polymerization, the stability of microtubules, and the incorporation of the microtubule-associated proteins (MAP2) and tau into microtubules. In vivo studies using Swiss 3T3 fibroblasts injected with YL 1/2 show that; when injected at low concentration (2 mg IgG/ml in the injection solution), the antibody binds to microtubules without changing their distribution in the cytoplasm. Injection of larger concentration of YL 1/2 (6 mg IgG/ml) induces the formation of microtubule bundles, and still higher concentrations cause the aggregation of microtubule bundles around the nucleus (greater than 12 mg IgG/ml).

Evaluation of immunotoxins containing single-chain ribosome-inactivating proteins and an anti-CD22 monoclonal antibody (OM124): in vitro and in vivo studies

1998 ◽  
Vol 101 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Andrea Bolognesi ◽  
Pier Luigi Tazzari ◽  
Fabiola Olivieri ◽  
Letizia Polito ◽  
Roberto Lemoli ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
In Vivo Studies ◽  
Single Chain ◽  
Ribosome Inactivating Proteins

An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor Stimulates Megakaryocytopoiesis

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 1981-1988
Author(s):  
Bijia Deng ◽  
Naheed Banu ◽  
Beth Malloy ◽  
Philip Hass ◽  
Jian Feng Wang ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Murine Monoclonal Antibody ◽  
In Vivo Studies ◽  
Severe Thrombocytopenia ◽  
Colony Forming Unit ◽  
Liquid Culture System ◽  
Primary Bone ◽  
American Society

Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34+ CD41+cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia. © 1998 by The American Society of Hematology.

Significance of a common epitope of plant and animal endomembranes

1986 ◽  
Vol 82 (1) ◽  
pp. 187-201
Author(s):  
G.P. Bolwell
Keyword(s):  
Monoclonal Antibody ◽  
Endoplasmic Reticulum ◽  
Membrane Trafficking ◽  
Animal Species ◽  
Antigenic Site ◽  
The Other ◽  
Fusion Event ◽  
The Common

A rat monoclonal antibody that was raised against a common epitope on bean (Phaseolus vulgaris) endomembranes has been shown to cross-react with microsomal polypeptides from a number of plant and animal species. Immunoblotting has shown that the epitope is present on a large subset of polypeptides on microsomes of five animal species. The antigenic site appears to be accessible on intact bean membranes since it is readily digested by trypsin. The epitope is probably not derived post-translationally since the same Mr range is immunoprecipitated from polypeptides newly synthesized in vivo and in vitro. The polypeptides in bean appear to be regulated independently, one of Mr 58,000, in particular, was highly induced by treatment of suspension cultures with fungal elicitor. Preincubation of membranes enriched with endoplasmic reticulum and Golgi apparatus with the antibody blocks transfer of radioactivity from one compartment to the other in vitro. The common antigenic site could possibly be concerned in recognition or some fusion event during membrane trafficking within the cell.

scholarly journals Morphological, Functional and Biochemical Characterization of Canine Gingival Fibroblasts

2013 ◽  
Vol 24 (2) ◽  
pp. 128-135 ◽  
Author(s):  
Camila Bonvicino Pelegrini ◽  
Luciana Prado Maia ◽  
Sérgio Luís Scombatti de Souza ◽  
Mário Taba Jr ◽  
Daniela Bazan Palioto
Keyword(s):  
Protein Content ◽  
Total Protein ◽  
Biochemical Analysis ◽  
Biochemical Characterization ◽  
In Vivo Studies ◽  
Total Protein Content ◽  
Gingival Fibroblasts

As dogs are good models for in vivo studies, it is interesting to evaluate the behavior of canine gingival fibroblasts (CGF) in vitro, so that these cells could be seeded on a matrix and later studied in vivo. The aim of this study was to perform a morphological, functional and biochemical analysis of CGF, comparing it with human gingival fibroblasts (HGF), as well as to evaluate the change of their characteristics over several passages. Using gingival fibroblasts from 3 dogs and 3 humans in the subculture (Sub), first (P1), third (P3), fifth (P5) and seventh (P7) passages, the following parameters were assessed: cell morphology, spreading, adhesion, viability and total protein content. The results showed no major differences between the passages in terms of morphology and spreading, and a tendency of greater adhesion and viability for HGF when compared with CGF. The total protein content was significantly higher for HGF. HGF exhibited greater functional and biochemical activity in vitro compared to CGF. Higher numbers at Sub were observed for both CGF and HGF in all evaluated parameters. The differences do not prevent the use of CGF for tissue engineering, but its use seems to be more appropriate in the subculture or first passage.

In vivo and in vitro cultured mouse preimplantation embryos differ in their display of a teratocarcinoma cell surface antigen: possible binding of an oviduct factor

Development ◽  
1985 ◽  
Vol 88 (1) ◽  
pp. 55-69
Author(s):  
Stephen J. Gaunt
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Surface Antigen ◽  
Antigenic Site ◽  
Cell Surface Antigen ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Teratocarcinoma Cell

By use of a monoclonal antibody, 2B5, in indirect immunofluorescence experiments, it was found that both fertilized and unfertilized mouse eggs obtained directly from the oviduct commenced expression of a cell surface antigen at about 5h after ovulation. Surface labelling became intense by 16 h after ovulation and persisted over all blastomeres throughout preimplantation development. In contrast, embryos cultured in vitro did not show appearance of 2B5 antigen until about 48 h after ovulation, at which time they were at the 2- to 4-cell stage. Antigen expression in vitro commonly began on a single blastomere and did not appear consistently over all blastomeres until the 8-cell stage (72h after ovulation). Unfertilized eggs maintained for 72 h in culture did not acquire 2B5 antigen. It is postulated that the absence of 2B5 antigen on 1-cell eggs cultured in vitro may be due either to a failure of normal synthesis by eggs as a result of a deficiency in the culture medium, or alternatively, to absence of a soluble oviduct factor which carries the 2B5 antigen, and which normally becomes bound to the surface of eggs after ovulation. The second of these two possibilities was supported by egg transfer experiments which showed that unfertilized eggs within the oviduct became 2B5 antigenpositive even after their prior fixation in glutaraldehyde. By the 2- to 4-cell stage, however, embryos developed their own capacity for synthesis of 2B5 antigen-positive cell surface molecules. This synthesis was inhibited by tunicamycin, suggesting that the antigenic site involved the sugar component of glycoprotein. The range of tissues within the postimplantation embryo and adult reproductive tracts which labelled with 2B5 antibody was found to be very similar to that known for SSEA-1 monoclonal antibody (Solter & Knowles, 1978; Fox et al. 1981; Fox, Damjanov, Knowles & Solter, 1982), and as further evidence of a relationship between 2B5 and SSEA-1 antigens it was found that 125I SSEA-1 antibody could be blocked in its binding to teratocarcinoma cells by preincubation in 2B5 monoclonal antibody.

An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor Stimulates Megakaryocytopoiesis

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 1981-1988 ◽  
Author(s):  
Bijia Deng ◽  
Naheed Banu ◽  
Beth Malloy ◽  
Philip Hass ◽  
Jian Feng Wang ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Murine Monoclonal Antibody ◽  
In Vivo Studies ◽  
Severe Thrombocytopenia ◽  
Colony Forming Unit ◽  
Liquid Culture System ◽  
Primary Bone ◽  
American Society

Abstract Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34+ CD41+cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia. © 1998 by The American Society of Hematology.

scholarly journals A monoclonal antibody directed against quail tyrosine hydroxylase: description and use in immunocytochemical studies on differentiating neural crest cells.

1989 ◽  
Vol 37 (8) ◽  
pp. 1197-1205 ◽  
Author(s):  
M Fauquet ◽  
C Ziller
Keyword(s):  
Monoclonal Antibody ◽  
Tyrosine Hydroxylase ◽  
Neural Crest ◽  
Mammalian Species ◽  
Culture Conditions ◽  
Phenotypic Traits ◽  
Neuronal Markers ◽  
Toxin Binding

Catecholamine (CA) synthesis is one of the phenotypic traits expressed by some neural crest-derived cells in vivo and in vitro. In the present study, we have evidenced, in quail embryos, the expression of the first enzyme of CA metabolism, tyrosine hydroxylase (TOH), using a monoclonal antibody raised against the quail enzyme. This antibody also recognizes TOH from chick and pleurodele, but not from several mammalian species (rat, human). We have also investigated the extent to which TOH-positive cells, differentiated in neural crest cultures, express structural neuronal markers and display vasoactive intestinal polypeptide (VIP) and substance P (SP) immunoreactivity. Double-immunolabeling experiments show that, in vitro, half of the population of TOH-positive cells exhibits tetanus toxin binding sites but none of them are recognized by a neurofilament antibody. On the other hand, some TOH-positive cells contain VIP or SP. These observations suggest that under our culture conditions autonomic neural crest precursors differentiate only into immature sympathoblasts, but are able to synthesize peptides in addition to CA.

scholarly journals Peptide Platform as a Powerful Tool in the Fight against COVID-19

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1667
Author(s):  
Michela Murdocca ◽  
Gennaro Citro ◽  
Isabella Romeo ◽  
Antonio Lupia ◽  
Shane Miersch ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Antigenic Site ◽  
Protein Docking ◽  
In Vivo Studies ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Dipeptidyl Peptidase 4 ◽  
Global Pandemic

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic causing over 195 million infections and more than 4 million fatalities as of July 2021.To date, it has been demonstrated that a number of mutations in the spike glycoprotein (S protein) of SARS-CoV-2 variants of concern abrogate or reduce the neutralization potency of several therapeutic antibodies and vaccine-elicited antibodies. Therefore, the development of additional vaccine platforms with improved supply and logistic profile remains a pressing need. In this work, we have validated the applicability of a peptide-based strategy focused on a preventive as well as a therapeutic purpose. On the basis of the involvement of the dipeptidyl peptidase 4 (DPP4), in addition to the angiotensin converting enzyme 2 (ACE2) receptor in the mechanism of virus entry, we analyzed peptides bearing DPP4 sequences by protein–protein docking and assessed their ability to block pseudovirus infection in vitro. In parallel, we have selected and synthetized peptide sequences located within the highly conserved receptor-binding domain (RBD) of the S protein, and we found that RBD-based vaccines could better promote elicitation of high titers of neutralizing antibodies specific against the regions of interest, as confirmed by immunoinformatic methodologies and in vivo studies. These findings unveil a key antigenic site targeted by broadly neutralizing antibodies and pave the way to the design of pan-coronavirus vaccines.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

1993 ◽  
Vol 51 ◽  
pp. 74-75
Author(s):  
Jason R. Swedlow ◽  
Neil Osheroff ◽  
Tim Karr ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Topoisomerase Ii ◽  
Biochemical Characterization ◽  
Developmental Cycle ◽  
Dna Topoisomerase Ii ◽  
Chromosomal Distribution ◽  
Dna Topoisomerase ◽  
Ideal System ◽  
Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

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