Membrane changes in neural target cells studied with fluorescent lectin probes

Development ◽  
1983 ◽  
Vol 77 (1) ◽  
pp. 183-200
Author(s):  
L. Gualandris ◽  
P. Rougé ◽  
A. M. Duprat
Keyword(s):  
Extracellular Matrix ◽  
Neural Induction ◽  
Internal Surface ◽  
Target Cells ◽  
Molecular Configuration ◽  
Lectin Receptors ◽  
Molecular Arrangement ◽  
Cell Layers ◽  
Pleurodeles Waltl

The competent ectoderm of Pleurodeles waltl comprises two cell layers with characteristic differences in their morphology, their composition and the molecular arrangement of the various constituents. The use of labelled lectin probes for observations of ectoderm tissue in vitro with u.v. microscopy (epi-illumination) and the quantification of the results show the following:- 1) Differences in labelling according to the nature of the lectins (SB A, PSA, LCA and Con A). These differences provide information on the nature of the carbohydrates which are present at this stage and on the number of receptors. 2) Differences in fluorescence intensity of the surfaces studied. The internal surface of the ectoderm is labelled more densely than the external surface. 3) Rearrangement of the lectin receptors with a new molecular configuration, stressing the fluidity of the membrane (by the mobility of the receptors throughout the membrane) and its importance for the occurrence of neural induction. 4) Existence of membrane glycoconjugate turnover. 5) A difference in behavioural characteristics between the internal and the external surfaces with respect to the lectins and the formation of an extracellular matrix on the internal surface alone. The extracellular matrix seems to have a role in morphogenetic movements.

Neural induction and the structure of the target cell surface

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 171-187
Author(s):  
A. M. Duprat ◽  
L. Gualandris ◽  
P. Rouge
Keyword(s):  
Cell Surface ◽  
Structural Integrity ◽  
Neural Induction ◽  
Active Role ◽  
Target Cells ◽  
Similar Treatment ◽  
Structural Modifications ◽  
Pleurodeles Waltl

Lectins (SBA and PSA) were used to provoke crowding and structural modifications of the presumptive ectoderm cell surface in order to investigate the role of the membrane organization of the competent target cells in neural induction. Are specific characteristics of the cell surface essential for this phenomenon to occur? From amphibian gastrulae, it is possible to obtain neural induction in vitro by association of presumptive ectoderm (target cells) with chordamesoderm (inductor tissue): 4 h of contact is sufficient in Pleurodeles waltl for transmission of the inductive signal. Very quickly, the treatment of the normal ectoderm by lectins (SBA-FITC or PSA-FITC) provoked surface modifications. Lectin-treatment (50 µg ml1−, 30 min) of presumptive ectoderm did not result in any neural induction. Lectin-treatment (50 µg ml1−, 30 min) of presumptive ectoderm previous to its association with the natural inductor for 4 h, disturbed the phenomenon: no induction. Similar treatment followed by association with the inductor for 24 h: induction. Treatment of SBA or PSA with their respective hapten inhibitors prior to addition to ectodermal cells completely blocked the suppressive effects on induction. The structural integrity of the membrane of competent target cells is necessary for neural induction to occur. The cell membrane could thus play, directly or indirectly, an active role in the specificity of this process

Neural induction: embryonic determination elicits full expression of specific neuronal traits

Development ◽  
1985 ◽  
Vol 89 (Supplement) ◽  
pp. 167-183
Author(s):  
A. M. Duprat ◽  
P. Kan ◽  
L. Gualandris ◽  
F. Foulquier ◽  
J. Marty ◽  
...  
Keyword(s):  
Neural Induction ◽  
Morphological Differentiation ◽  
Target Cells ◽  
Adhesive Properties ◽  
Neuronal Markers ◽  
Toxin Binding ◽  
Neurofilament Polypeptides ◽  
Pleurodeles Waltl ◽  
High Degree

In Pleurodeles waltl, the early neuronal differentiation of precursor cells from late gastrula stage has been studied by culture in vitro from either isolated neural plate (NP) or isolated neural fold (NF). The aim of this study was to delineate the information acquired by ectodermal target cells during neural induction. By culturing these cells in vitro either with or without the underlying chordamesoderm, we showed that in the absence of chordamesodermal influence such NP or NF cells exhibited a high degree of biochemical and morphological differentiation as revealed by the synthesis and the storage of neurotransmitters, the activity of specific enzymes, as well as by the expression of neuronal markers: specific changes in cell surface carbohydrates, tetanus toxin binding sites and neurofilament polypeptides. Remarkable changes in the cell adhesive properties were the first events observed in the different central (NP) and peripheral (NF) types. In cocultures the chordamesodermal cells exert a beneficial influence on this differentiation, specially increasing acetylcholine synthesis. There are some differences between central (NP) or peripheral (NF) neuroblast response to this further notochord or mesodermal influence.

Expression of N-CAM precedes neural induction in Pleurodeles waltl (urodele, amphibian)

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 675-683 ◽  
Author(s):  
J.P. Saint-Jeannet ◽  
F. Foulquier ◽  
C. Goridis ◽  
A.M. Duprat
Keyword(s):  
Monoclonal Antibody ◽  
Nervous System ◽  
Xenopus Laevis ◽  
Neural Induction ◽  
Gastrula Stage ◽  
Pleurodeles Waltl ◽  
Early Gastrula

The appearance and localization of N-CAM during neural induction were studied in Pleurodeles waltl embryos and compared with recent contradictory results reported in Xenopus laevis. A monoclonal antibody raised against mouse N-CAM was used. In the nervous system of Pleurodeles, it recognized two glycoproteins of 180 and 140×10(3) M(r) which are the Pleurodeles equivalent of N-CAM-180 and -140. Using this probe for immunohistochemistry and immunocytochemistry, we showed that N-CAM was already expressed in presumptive ectoderm at the early gastrula stage. In late gastrula embryos, a slight increase in staining was observed in the neurectoderm, whereas the labelling persisted in the noninduced ectoderm. When induced ectodermal cells were isolated at the late gastrula stage and cultured in vitro up to 14 days, a faint polarized labelling of cells was observed initially. During differentiation, the staining increased and became progressively restricted to differentiating neurons.

scholarly journals Characterization of human marrow stromal cells: role in progenitor cell binding and granulopoiesis

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1794-1800
Author(s):  
JL Liesveld ◽  
CN Abboud ◽  
RE Duerst ◽  
DH Ryan ◽  
JK Brennan ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Horse Serum ◽  
Ldl Receptor ◽  
Adherent Cell ◽  
Target Cells ◽  
Cell Binding ◽  
Cell Layers ◽  
Vitro Model

Adherent cell layers and their associated extracellular matrices form when human marrow is incubated in cultures containing hydrocortisone and horse serum. These stromal layers contain cells positive for alkaline phosphatase; secrete collagens types I and III and fibronectin, bind the anti-actin monoclonal antibodies (MoAbs) HHF and CGA-7; stain with oil red O, and express the acetylated LDL receptor. Highly purified CD34 (My10)-positive progenitor cells attach to these stromal layers, and a 16-fold enrichment of CFU-GM in both stromal attachment and semisolid agar assays was observed. Granulopoiesis persisted up to 40 days (mean duration 25 days) after passaged stroma were recharged with stromal cell-depleted target cells in a two-stage liquid marrow culture system. Although equal to marrow fibroblasts in their ability to bind CD34+ myeloid progenitors, stromal layers were better at supporting granulopoiesis. This system provides an in vitro model to characterize the components of stroma and stroma-cytomatrix that enhance marrow progenitor cell localization and maintenance.

scholarly journals Preclinical and Toxicology Studies of BRD5529, a Selective Inhibitor of CARD9

2021 ◽  
Author(s):  
Theodore Kottom ◽  
Kyle Schaefbauer ◽  
Eva M Carmona ◽  
Joanne E Yi ◽  
Andrew Limper
Keyword(s):  
Extracellular Matrix ◽  
Blood Chemistry ◽  
Pneumocystis Pneumonia ◽  
Tnf Alpha ◽  
Blood Collection ◽  
Type I ◽  
Beta Glucan ◽  
Lectin Receptors ◽  
Liver And Kidney

Background: Exuberant inflammation during Pneumocystis pneumonia leads to lung injury. CARD9 is a central mediator of inflammatory signaling mediated by C-type lectin receptors. CARD9 inhibitor BRD5529 has been shown to be an effective in vitro inhibitor of Pneumocystis Beta-glucan-induced proinflammatory signaling and downstream TNF-alpha production, suggesting its viability as a candidate for preliminary drug testing as an anti-inflammatory agent in the rodent Pneumocystis pneumonia model (PCP). Methods: To assess for potential toxicity, mice were injected intraperitoneally (IP) daily either with vehicle or BRD5529 at 0.1 mg/kg or 1.0 mg/kg for two weeks. Mouse weights were taken daily. At day 14, mice were euthanized, weighed, and analyzed by flexiVent for lung stiffness. Lungs, liver, and kidney were then harvested for H&E staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines via ELISA and extracellular matrix generation via quantitative PCR (q-PCR). Blood collection postmortem was performed for blood chemistry analysis. Results: BRD5529 at both doses of IP administration resulted in no significant changes in daily or final weight gain. Analysis of lung stiffness by flexiVent showed no significant differences between the control or treated groups. Furthermore, ELISA results for proinflammatory IL-1 Beta, IL-6, and TNF-alpha showed no major differences in the respective groups. qPCR analysis of extracellular matrix transcripts collagen type I, alpha 1 (Col1a1) and fibronectin (Fn) were statically similar as well in the treated and control groups. Examination and pathology scoring of H&E slides from lung, liver, and kidney from the each of the mice in all groups and subsequent pathology scoring showed no significant change. Blood chemistry analysis revealed similar, non-significant patterns. Conclusions: BRD5529 in our initial general safety and toxicology assessments displayed no inherent safety concerns in the analyzed parameters. These data support broader in vivo testing of the inhibitor as a timed adjunct therapy to the deleterious proinflammatory host immune response often associated with anti-Pneumocystis therapy.

scholarly journals Characterization of human marrow stromal cells: role in progenitor cell binding and granulopoiesis

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1794-1800 ◽  
Author(s):  
JL Liesveld ◽  
CN Abboud ◽  
RE Duerst ◽  
DH Ryan ◽  
JK Brennan ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Horse Serum ◽  
Ldl Receptor ◽  
Adherent Cell ◽  
Target Cells ◽  
Cell Binding ◽  
Cell Layers ◽  
Vitro Model

Abstract Adherent cell layers and their associated extracellular matrices form when human marrow is incubated in cultures containing hydrocortisone and horse serum. These stromal layers contain cells positive for alkaline phosphatase; secrete collagens types I and III and fibronectin, bind the anti-actin monoclonal antibodies (MoAbs) HHF and CGA-7; stain with oil red O, and express the acetylated LDL receptor. Highly purified CD34 (My10)-positive progenitor cells attach to these stromal layers, and a 16-fold enrichment of CFU-GM in both stromal attachment and semisolid agar assays was observed. Granulopoiesis persisted up to 40 days (mean duration 25 days) after passaged stroma were recharged with stromal cell-depleted target cells in a two-stage liquid marrow culture system. Although equal to marrow fibroblasts in their ability to bind CD34+ myeloid progenitors, stromal layers were better at supporting granulopoiesis. This system provides an in vitro model to characterize the components of stroma and stroma-cytomatrix that enhance marrow progenitor cell localization and maintenance.

Expression of tenascin in response to neural induction in amphibian embryos

Development ◽  
1988 ◽  
Vol 104 (3) ◽  
pp. 511-524 ◽  
Author(s):  
J.-F. Riou ◽  
D.-L. Shi ◽  
M. Chiquet ◽  
J.-C. Boucaut
Keyword(s):  
Cell Migration ◽  
Neural Crest Cell ◽  
Neural Induction ◽  
Gastrula Stage ◽  
Directed Cell Migration ◽  
Pleurodeles Waltl ◽  
Migration Pathways ◽  
Early Gastrula

The expression of tenascin, a constituent of extracellular matrix (ECM), was studied during the embryonic development of the amphibian Pleurodeles waltl. An antiserum to chick fibroblast tenascin was shown to cross-react with the homologous molecule of the amphibian. Immunostaining of embryo sections with anti-tenascin antiserum revealed that tenascin appears just after the completion of neurulation. At the tailbud stage, tenascin is present in the ECM located at sites of directed cell migration (neural crest cell migration pathways, extension of the pronephretic duct) and mesenchyme condensation (endocardium, aortic arches). The accumulation of tenascin immunoreactivity in the embryonic ECM is correlated with the synthesis of the 220×103Mr polypeptide of the molecule. To provide data on the patterning of tenascin, ectoderm and dorsal blastoporal lip isolated at early gastrula stage were cultured for a period of 3 days. Epidermal vesicles differentiating from isolated ectoderm completely lack tenascin. Conversely, axial mesoderm derivatives present in cultured dorsal blastoporal lip were found to produce tenascin. Neural induction of ectoderm isolated at early gastrula stage was performed in vitro with the dorsal blastoporal lip or concanavalin A. The induced neural tissue was found to accumulate tenascin. Spemann experiments confirmed in vivo that tenascin is expressed by ectodermal cells as a response to neural induction.

scholarly journals Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix.

1982 ◽  
Vol 92 (2) ◽  
pp. 485-492 ◽  
Author(s):  
J A McDonald ◽  
D G Kelley ◽  
T J Broekelmann
Keyword(s):  
Extracellular Matrix ◽  
Binding Domain ◽  
Collagen Deposition ◽  
Domain Specific ◽  
Collagen Organization ◽  
Cell Layers ◽  
Fibrillar Component ◽  
Fibronectin Deposition

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti-60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.

Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

1981 ◽  
Vol 39 ◽  
pp. 452-453
Author(s):  
K. E. Muse ◽  
D. G. Fischer ◽  
H. S. Koren
Keyword(s):  
Target Cell ◽  
Human Peripheral Blood ◽  
Mononuclear Phagocytes ◽  
Ultrastructural Changes ◽  
Target Cells ◽  
Limited Information ◽  
Blood Monocytes ◽  
Tumoricidal Activity ◽  
Activated State

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

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