Rüdiger Schade

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c‐Jun and p53 with consequence for their ubiquitin (Ub)‐dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co‐purify with CSN. Immunoprecipi tation and far‐western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold‐labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co‐immuno precipitates with CSN from HeLa cells. CK2 binds to ΔCSN3(111–403) and CSN7, whereas PKD interacts with full‐length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c‐Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN‐associated kinases and induce degradation of c‐Jun in HeLa cells. Curcumin treatment results in elevated amounts of c‐Jun–Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.

Use of Polyclonal Avian Antibodies

for many years antibodies have been used for quantitation and detection of a lot of different substances. The specific recognition between antibodies and antigens has led to the development of diverse methods for basic research in the sciences as well as for diagnosis in human and veterinary medicine.

Isolation of IgY from Yolk

The yolk in hens’ eggs consists of dry matter (51,3 %) and water (48,7 %) in nearly equal proportions (Siewert and Bronch, Handbuch der Tierernahrung, 1972; Romanoff und Romanoff, The Avian Egg, 1949). The dry matter is made up as shown in Table 1.

Short Introduction to Hens’ Humoral Immune System

The hens’ immune system differs from mammals’ in various ways. In this brief introduction we shall try to give a basic idea of the special features of the hens’ humoral immune system, especially as regards the structures and functions of antibodies.

Possibilities of Keeping Laying-Hens to Obtain Antibodies

Within the field of biological-medical research, laying-hens are being kept increasingly for experimental purposes, but they have a different status to most other laboratory animals in counting as farmyard-animals. There are no defined regulations as regards hens for the laboratory but there are basic regulations for the protection of animals as well as national and European regulations, guidelines and recommendations for hen-keeping for experiments:

Pros and Cons of Obtaining Antibodies from Chickens’ Eggs Rather than from Mammals’ Serum

The fact that IgY-technology lessens the pain of animals used in experiments should be reason enough for adopting it, but unfortunately the first consideration is generally the cost.

Production, purification, and characterization of rat pro-CCK from serum-free adapted Drosophila cells.

The precursor of cholecystokinin (pro-CCK) was expressed and purified from media of stably transfected D.Mel-2 cell as an V5-His tagged fusion protein. Its identity was confirmed using SDS-PAGE, immunoblotting, gel filtration chromatography, HPLC, and Mass Spectroscopy. Two major forms of pro-CCK were found with a molecular weight of about 14.4 and 11.3 kDa. The smaller form represents the V5-His tagged pro-CCK after cleavage at a single arginine residue at CCK-58. This cleavage is probably being performed by endogenous proteases in these cells. Purification of the desired larger form of pro-CCK is possible using a nickel column with a recovery of about 20%, yielding 500 microg/L media. The purified protein is stable for several months and can be used for further functional studies of pro-CCK.

IgY-Antibodies, Future Prospects

In the past two decades, biomedicai research has been confronted with a growing public interest in the welfare of animals used in research. The resulting discussion has encouraged the search for ways to refine, reduce or even replace animal experiments. This also applies to the production of mono- and polyclonal antibodies in animals. These antibodies are important tools in bio-medical research, e.g. they are essential components of diagnostic methods for quantitative and qualitative determi-nations of a wide variety of biological molecules. Polyclonal antibodies are produced in rabbits and other rodents like mice, rats and guinea-pigs or in farm animals such as horses, sheep and goats. The production of polyclonal antibodies involves two stressful procedures, firstly the immunisation pro-cedure with several injections and secondly the harvesting of the antibodies which involves bleeding of the animals.