Alterations in membrane polypeptides of chick embryo fibroblasts induced by transformation with avian sarcoma viruses.

Membrane proteins of chick embryo fibroblasts (CEF) transformed with various strains of avian sarcoma viruses were analyzed by electrophoresis in SDS-polyacrylamide gels and compared with those of untransformed cells. The following differences were consistently detected in CEF transformed with B77, the Prague strain of Rous sarcoma virus (PR-RSV) or the Schmidt-Ruppin strain of RSV (SR-RSV): (1) The appearance of a polypeptide band with an apparent molecular weight of 90,000, (2) increase in amount of a polypeptide of 79,000 daltons, (3) significant decrease in amount of a polypeptide of 50,000 daltons and (4) marked decrease in amount of a protein of 200,000 daltons. CEF infected with the temperature-sensitive (ts) mutants of these strains, LA334 (of B77), LA31 (of PR-RSV) or OS122 (of SR-RSV) showed similar changes at 36°, but at 41°, except for alteration (4), the profiles of the membrane proteins were similar to those of uninfected cells. Changes (1) and (3) were reversible and clearly observable within a few hours after a temperature shift of CEF infected with ts mutants. Fusiform transformation induced by a variant of B77 was also shown to induce alterations (1) and (3).From these and other results, the appearance of the polypeptide band of 90,000 daltons, which could not be detected in untransformed cells, and the marked decrease in amount of a protein of 50,000 daltons in cell membranes were concluded to be closely correlated with transformation of CEF.     

The use of the chorio-allantoic membrane of the chick embryo as test for anti-inflammatory activity

Inflammation Research -     

Acute inflammatory pulmonary reactions induced by chemotactic factors.

Acute inflammatory reactions have been produced in hamster lungs by the intrapulmonary instillation of preformed chemotactic mediators (C5fr and F-Met-Leu-Phe). By the use of 111Indium and 125Iodine labeling of homologous neutrophils (PMNs) and homologous albumin, respectively, it has been possible to obtain highly reproducible and quantitative parameters of the acute inflammatory response. The lung responds in a dose-dependent and time-dependent manner to the instillation of preformed chemotactic mediators. The quantitative parameters indicative of PMN influx were more prominent than changes in vascular permeability. The data obtained by the use of radiolabeled PMNs were confirmed by observation with light microscopy. Nonchemotactic substances such as human IgG, serum albumin, and C4 failed to induce inflammatory responses in lung. Interestingly, intact C5 instilled into lung was extremely phlogistic, apparently due to hydrolysis once within the lung. These studies provide an approach to reliable quantitative parameters of inflammatory reactions in the lung and emphasize the in vivo biologic effects of chemotactic mediators.     

Mechanism of the induction of angiogenesis by human neoplastic lymphoid tissue: studies on the chorioallantoic membrane (CAM) of the chick embryo

The angiogenic activity of various neoplastic and control tissues, cells and extracts has been tested on the chorioallantoic membrane of the chick (CAM). The vascular response was assessed macroscopically and also by histological examination. Angiogenesis was induced by a number of neoplastic implants the most potent being derived from Hodgkin's disease, histiocytic lymphoma or glioma tissue. Boiled tumour tissue was ineffective. Lymphocytes extracted from human lymphomas, activated normal peripheral blood lymphocytes and established lymphoid cell lines of neoplastic origin were generally effective in inducing neovascularisation through millipore membranes as were 90,000-100,000 MW fractions of human tumour tissue. In all cases examined histologically a mononuclear cell infiltrate in the CAM mesoderm accompanied a positive vascular response. These results implicate host monocytes in the generation of neovascularisation by neoplastic tissue.     

The chick embryo appears as a natural model for research in beta-amyloid precursor protein processing

This study reveals that the chick embryo has active the machinery for the production and degradation of the amyloid beta peptide characteristic of Alzheimer's disease. We cloned the principal beta-amyloid precursor protein isoforms in the chick embryo and observed that they are highly homologous to the human sequences and identical at the C-terminal sequence, including the amyloid beta domain. Mammals such as rat or mouse, more commonly used as animal models of human diseases, have a distinct amyloid beta sequence. The distribution of beta-amyloid precursor protein isoforms in the chick embryo revealed that, as in humans, their expression is ubiquitous and the prototype beta-amyloid precursor protein-695 predominated in the nervous system. We also found that the chick embryo expresses the genes for the main proteolytic proteases implicated in the production of amyloid beta, including BACE-1, BACE-2, presenilin-1, presenilin-2 and nicastrin, as well as the amyloid beta-degrading enzyme neprilysin, or ADAM-17, a protease implicated in the non-amyloidogenic processing of beta-amyloid precursor protein. We have also found that between amyloid beta40 and amyloid beta42, this latter seems to be the major amyloid beta peptide produced during chick embryogenesis. The chick embryo appears as a suitable natural model to study cell biology and developmental function of beta-amyloid precursor protein and a potential assay system for drugs that regulate beta-amyloid precursor protein processing.     

In vivo time-course of the angiogenic response induced by multiple myeloma plasma cells in the chick embryo chorioallantoic membrane

In this study, we set out to make a fine characterization of the angiogenic response induced by plasma cells obtained from patients with active-multiple myeloma (MM), in comparison with cells obtained from patients with non-active MM and benign lesions such as monoclonal gammopathy of undetermined significance (MGUS), in the chick embryo chorioallantoic membrane (CAM) assay. To achieve this we investigated the time-course of the angiogenic response induced by gelatin sponges soaked in the cell suspensions and implanted on the CAM surface from day 8 to day 12 of incubation by evaluating the number of vessels, of the vessel bifurcation and the intervascular distance at 24, 48, 72 and 96 h after the implants. The results show that plasma cell suspensions obtained from patients with active MM induce a vasoproliferative response that was significantly higher than that induced by cell suspensions obtained from patients with non-active MM or with MGUS, which is also a function of the day of implantation. In fact, implants made from day 8 to day 10 induce a strong angiogenic response, whereas those made from day 11 to day 12 do not. This finding might depend on the fact that CAM endothelium exhibits an intrinsically high mitotic rate until day 10. Thereafter, the endothelial mitotic index declines rapidly, and consequently cell suspensions implanted on the CAM of successively older embryos are not able to induce a vasoproliferative response in parallel with the reduced rates of growth of the CAM's endothelial cells.     

Pulmonary veins in the chick embryo: Origin as determined by radioautographic mapping

In the head process to early head fold stage chick embryo, the cells which will form the pulmonary veins are located in the mesoderm near the posteromedial edges of the heart-forming regions. At the 26 somite to early limb bud stage, the presumptive pulmonary vein cells have been folded to the midline of the embryo as part of the splanchnic mesoderm and form the endothelial plexus which courses through the dorsal mesentery of the sinoatrial region of the heart.     

Improved method for chick whole-embryo culture using a filter paper carrier.

We describe a simple method of chick whole-embryo culture, which uses a filter paper carrier to hold the early blastoderm and vitelline membranes under tension while the embryo grows on a substratum of agar-albumen. This is a quick and efficient means of setting up cultures of chick embryos beginning at pre-primitive streak stages to stage 10 (stages X--XIV, Eyal-Giladi and Kochav [1976] Dev Biol 49:321-337; stages 1--10, Hamburger and Hamilton [1951] J Morphol 88:49--92). This is an improvement on the original method of New, which used a glass ring and watch glass (New [1955] Exp Morphol 3:320--331). Our modification of New's method, which we call EC (Early Chick, pronounced EASY) culture, facilitates several manipulations in early chick embryos, including microsurgery, grafting, bead implantation, microinjection, and electroporation. Using the EC method, embryos at stage 8 and older can be readily cultured either dorsal-side up (in contrast to New's method) or ventral-side up, as desired; embryos younger than stage 8 can be culture only ventral-side up (as with New's method). We also discuss some alternative methods for setting up these cultures.     

The chick chorioallantoic membrane as a novel in vivo model for the testing of biomaterials

Current in vivo models for testing biomaterials are time and labor intensive as well as expensive. This article describes a new approach for testing biomaterials in vivo using the chorioallantoic membrane (CAM) of the developing chicken embryo, as an alternative to the traditional mammalian models. Fertilized chicken eggs were incubated for 4 days, at which time a small window was cut in the shell of the egg. After 1 week of incubation, the CAM received several test materials, including the endotoxin LPS, a cotton thread and a Silastic tubing. One day and 1 week later, the tissue response to the test materials was assessed using gross, histological, and scanning electron microscope evaluations. The inflammatory response of the chorioallantoic membrane to biomaterials was fully characterized and found to be similar to that of the mammalian response and was also seen to vary according to test materials. We also found that the structure and geometry of the test materials greatly influenced the incorporation of the samples in the CAM. The similarity of the tissue response of the CAM with the mammalian models, plus the low cost, simplicity, and possibility to continuously visualize the test site through the shell window make this animal model particularly attractive for the rapid in vivo screening of biomaterials.     

Tumor angiogenesis induced by granulocyte chemotactic protein-2 as a countercurrent principle

Chemokine production by tumors is a well-known phenomenon, but its role in tumor biology remains debatable. Although intratumoral injection of granulocyte chemotactic protein-2 (GCP-2) had no effect on tumor parameters, needle-free stable expression of the chemokine resulted in enhanced tumor growth. It is shown here that tumors that express a potent form of GCP-2 induce a strong influx and activation of tumor-associated neutrophils. The production of GCP-2 leads to intratumoral expression of gelatinase B and advantage for tumor growth by increased angiogenesis. These results are in line with the countercurrent principle of chemokine action and support the notion that paraneoplastic expression of ELR-positive CXC chemokines has to be blocked rather than stimulated in cancer therapy.     

Egg:embryo weight ratio as an indicator of dwarfism induced by infectious bronchitis virus.

A simple objective method to quantify embryo dwarfism induced by infectious bronchitis virus in embryonated chicken eggs has been used to determine endpoints in virus titration and neutralization assays. The eggs and the respective embryos were weighed and embryo:egg weight (EE) ratios were calculated. The EE ratios were compared with the uninoculated control eggs and endpoints could be calculated objectively. EE indices were also calculated by dividing the EE ratios of inoculated embryonated chicken eggs by the mean EE ratio of uninoculated controls, or in the case of virus neutralization tests by the mean EE ratio of eggs inoculated with virus alone. Although this mean EE index did not reflect the dwarfing (or lack of it) in individual eggs, it served as a group indicator. This method would be useful to observe embryo lesions especially in field (non-egg adapted) infectious bronchitis virus isolates, which does not cause observable dwarfing until several embryo passages.     

Eggshell membrane biomaterial as a platform for applications in materials science

Eggshell membrane (ESM) is a unique biomaterial, which is generally considered as waste. However, it has extraordinary properties which can be utilized in various fields and its potential applications are therefore now being widely studied. The first part of this review focuses on the chemical composition and morphology of ESM. The main areas of ESM application are discussed in the second part. These applications include its utilization as a biotemplate for the synthesis of nanoparticles; as a sorbent of heavy metals, organics, dyes, sulfonates and fluorides; as the main component of biosensors; in medicine; and various other applications. For each area of interest, a detailed literature survey is given.