scholarly journals Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes.

1992 ◽  
Vol 119 (2) ◽  
pp. 475-482 ◽  
Author(s):  
M A Moses ◽  
J Sudhalter ◽  
R Langer
Keyword(s):  
Growth Factor ◽  
Potent Inhibitor ◽  
Chorioallantoic Membrane ◽  
Boyden Chamber ◽  
Chick Chorioallantoic Membrane ◽  
Isolation And Characterization ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
And Migration

An inhibitor of neovascularization from the conditioned media of scapular chondrocytes established and maintained in serum-free culture has been isolated and characterized. To determine whether this chondrocyte-derived inhibitor (ChDI) was capable of inhibiting neovascularization in vivo, this protein was assayed in the chick chorioallantoic membrane assay. ChDI was a potent inhibitor of angiogenesis in vivo (4 micrograms = 87% avascular zones). This inhibitor is also an inhibitor of fibroblast growth factor-stimulated capillary endothelial cell (EC) proliferation and migration, as well as being an inhibitor of mammalian collagenase. ChDI significantly suppressed capillary EC proliferation in a dose-dependent, reversible manner with an IC50 (the inhibitory concentration at which 50% inhibition is achieved) of 2.025 micrograms/ml. Inhibition by ChDI of growth factor-stimulated capillary EC migration was also observed using a modified Boyden chamber assay (IC50 = 255 ng/ml). SDS-PAGE analysis followed by silver staining of ChDI purified to apparent homogeneity revealed a single band having an M(r) of 35,550. Gel elution experiments demonstrated that only protein eluting at this molecular weight was anti-angiogenic. These studies are the first demonstration that chondrocytes in culture can produce a highly enriched, potent inhibitor of neovascularization which also inhibits collagenase.

Transepithelial calcium transport in the chick chorioallantoic membrane. I. Isolation and characterization of chorionic ectoderm cells

1993 ◽  
Vol 105 (2) ◽  
pp. 369-379 ◽  
Author(s):  
R.E. Akins ◽  
R.S. Tuan
Keyword(s):  
Calcium Binding ◽  
Chorioallantoic Membrane ◽  
Experimental Manipulation ◽  
Transepithelial Transport ◽  
Temperature Sensitive ◽  
Specific Cell ◽  
Chick Chorioallantoic Membrane ◽  
Isolation And Characterization

The chicken eggshell supplies approximately 80% of the calcium found in the hatchling chick. The mobilization of eggshell calcium into the developing embryo involves the transepithelial transport of large amounts of calcium in a development-specific manner. The cells responsible for the transport of eggshell calcium into the embryonic circulation are the ectodermal cells of the chorioallantoic membrane. In this report, we present a method for the isolation and culture of chorioallantoic membrane ectodermal cells, which are amenable to direct experimental manipulation. Cell preparations are characterized with respect to the expression of an ectoderm-specific cell surface marker (transcalcin, a calcium-binding protein), and a specific enzymatic activity (elevated Ca(2+)-activated ATPase). Functional assessment of in vitro cellular calcium uptake by 45Ca2+ tracer kinetics indicates the persistence of a temperature-sensitive, rapid-influx pathway similar to that observed in vivo. The preparations of primary ectodermal cells present an in vitro system applicable to the experimental analysis of calcium metabolism and transport by the chick chorioallantoic membrane.

scholarly journals p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2–stimulated angiogenesis

2002 ◽  
Vol 156 (1) ◽  
pp. 149-160 ◽  
Author(s):  
Taro Matsumoto ◽  
Ingela Turesson ◽  
Majlis Book ◽  
Pär Gerwins ◽  
Lena Claesson-Welsh
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Vascular Endothelial Cells ◽  
Chorioallantoic Membrane ◽  
Collagen Gel ◽  
P38 Map Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Chick Chorioallantoic Membrane

The p38 mitogen–activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor–dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2–induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2–induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2–induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2–induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2–driven angiogenesis.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

The chick embryo chorioallantoic membrane model: a research approach for ex vivo and in vivo experiments

2021 ◽  
Vol 28 ◽  
Author(s):  
Ana Isabel Fraguas-Sánchez ◽  
Cristina Martín-Sabroso ◽  
Ana Isabel Torres-Suárez
Keyword(s):  
Animal Models ◽  
Chorioallantoic Membrane ◽  
New Drugs ◽  
Biological Research ◽  
Research Areas ◽  
Chick Chorioallantoic Membrane ◽  
Biodistribution Studies ◽  
Toxicological Studies ◽  
Cam Model

Background: The chick chorioallantoic membrane (CAM) model has attracted a great deal of interest in pharmaceutical and biological research as an alternative or complementary in vivo assay to animal models. Traditionally, CAM assay has been widely used to perform some toxicological studies, specifically to evaluate the skin, ocular and embryo toxicity of new drugs and formulations, and perform angiogenesis studies. Due to the possibility to generate the tumors onto the CAM, this model has also become an excellent strategy to evaluate the metastatic potential of different tumours and test the efficacy of novel anticancer therapies in vivo. Moreover, in the recent years, its use has considerably grown in other research areas, including the evaluation of new anti-infective agents, the development of biodistribution studies and tissue engineering research. Objectives: This manuscript provides a critical overview of the use of CAM model in pharmaceutical and biological research, especially to test the toxicity of new drugs and formulations and the biodistribution and the efficacy of novel anticancer and anti-infective therapies, analyzing its advantages and disadvantages compared to animal models. Conclusion: The chick chorioallantoic membrane model shows great utility in several research areas, such as cancer, toxicology, biodistribution studies and anti-infective therapies. In fact, it has become an intermediate stage between in vitro experiments and animal studies, and, in the case of toxicological studies (skin and ocular toxicity), has even replaced the animal models.

scholarly journals ELECTRON PROBE ANALYSIS OF THE CALCIUM DISTRIBUTION IN CELLS OF THE EMBRYONIC CHICK CHORIOALLANTOIC MEMBRANE I. A CRITICAL EVALUATION OF TECHNIQUES

1972 ◽  
Vol 20 (6) ◽  
pp. 401-413 ◽  
Author(s):  
JAMES R. COLEMAN ◽  
A. RAYMOND TEREPKA
Keyword(s):  
Electron Probe ◽  
Critical Evaluation ◽  
Chorioallantoic Membrane ◽  
Embryonic Chick ◽  
Total Calcium ◽  
Electron Probe Analysis ◽  
X Ray ◽  
Chick Chorioallantoic Membrane ◽  
Minor Losses

The chorioallantoic membrane of the developing chick embryo is an epithelium that actively transports calcium. The methodology utilized to prepare this soft tissue for calcium localization with the electron probe x-ray microanalyzer is presented in detail. The preparative procedures are evaluated according to general histochemical principles and in relationship specifically to electron probe investigations. It is shown that the method employed in these studies preserves the normal fine structure of the tissue, prevents selective loss of calcium, permits only minor losses of total calcium and appears to maintain the distribution of calcium that existed in vivo. Examples are presented of artifacts that can be induced during tissue sectioning and mounting procedures. Problems of defining electron probe resolution in biologic specimens are discussed, and the critical importance of evaluating x-ray images in association with simultaneously generated sample current images is emphasized.

scholarly journals Gypenoside LXXV Promotes Cutaneous Wound Healing In Vivo by Enhancing Connective Tissue Growth Factor Levels Via the Glucocorticoid Receptor Pathway

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1595 ◽  
Author(s):  
Sungjoo Park ◽  
Eunsu Ko ◽  
Jun Hyoung Lee ◽  
Yoseb Song ◽  
Chang-Hao Cui ◽  
...  
Keyword(s):  
Wound Healing ◽  
Growth Factor ◽  
Connective Tissue ◽  
Wound Closure ◽  
Tissue Growth ◽  
Cutaneous Wound Healing ◽  
Cutaneous Wound ◽  
And Migration ◽  
Proliferation And Migration

Cutaneous wound healing is a well-orchestrated event in which many types of cells and growth factors are involved in restoring the barrier function of skin. In order to identify whether ginsenosides, the main active components of Panax ginseng, promote wound healing, the proliferation and migration activities of 15 different ginsenosides were tested by MTT assay and scratched wound closure assay. Among ginsenosides, gypenoside LXXV (G75) showed the most potent wound healing effects. Thus, this study aimed to investigate the effects of G75 on wound healing in vivo and characterize associated molecular changes. G75 significantly increased proliferation and migration of keratinocytes and fibroblasts, and promoted wound closure in an excision wound mouse model compared with madecassoside (MA), which has been used to treat wounds. Additionally, RNA sequencing data revealed G75-mediated significant upregulation of connective tissue growth factor (CTGF), which is known to be produced via the glucocorticoid receptor (GR) pathway. Consistently, the increase in production of CTGF was confirmed by western blot and ELISA. In addition, GR-competitive binding assay and GR translocation assay results demonstrated that G75 can be bound to GR and translocated into the nucleus. These results demonstrated that G75 is a newly identified effective component in wound healing.

scholarly journals Silencing of MBD1 and MeCP2 in prostate-cancer-derived PC3 cells produces differential gene expression profiles and cellular phenotypes

2008 ◽  
Vol 28 (6) ◽  
pp. 319-326 ◽  
Author(s):  
Ahmed Yaqinuddin ◽  
Farhat Abbas ◽  
Syed Z. Naqvi ◽  
Mohammad U. Bashir ◽  
Romena Qazi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Mrna Expression ◽  
Cellular Proliferation ◽  
Expression Profiles ◽  
Boyden Chamber ◽  
Invasion And Migration ◽  
Pc3 Cells ◽  
And Migration

Alterations in genomic CpG methylation patterns have been found to be associated with cell transformation and neoplasia. Although it is recognized that methylation of CpG residues negatively regulates gene expression, how the various MBPs (methyl-binding proteins) contribute to this process remains elusive. To determine whether the two well characterized proteins MeCP2 (methyl-CpG-binding protein 2) and MBD1 (methyl-CpG-binding domain 1) have distinct or redundant functions, we employed RNAi (RNA interference) to silence their expression in the prostate cancer-derived PC3 cell line, and subsequently compared cell growth, invasion and migration properties of these cell lines in addition to their respective mRNA-expression profiles. Cells devoid of MeCP2 proliferated more poorly compared with MBD1-deficient cells and the parental PC3 cells. Enhanced apoptosis was observed in MeCP2-deficient cells, whereas apoptosis in parental and MBD1-deficient cells appeared to be equivalent. Boyden chamber invasion and wound-healing migration assays showed that MBD1-silenced cells were both more invasive and migratory compared with MeCP2-silenced cells. Finally, gene chip microarray analyses showed striking differences in the mRNA-expression profiles obtained from MeCP2- and MBD1-depleted cells relative to each other as well as when compared with control cells. The results of the present study suggest that MeCP2 and MBD1 silencing appear to affect cellular processes independently in vivo and that discrete sets of genes involved in cellular proliferation, apoptosis, invasion and migration are targeted by each protein.

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