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Cross-linking of cell surface receptors by multivalent ligands, e.g. by antibodies, evokes their clustering - patching. Subsequently, these clusters can be translocated by the acto-myosin machinery toward one pole of the cell and assembly cap. Patching of FcγRII in U937 cells correlates with tyrosine phosphorylation of several proteins while cap assembly correlates with their dephosphorylation. To study the mechanism of activation of tyrosine kinases during FcγRII activation we disturbed the organization of the putative plasma membrane microdomains by depletion of membrane cholesterol and sphingomyelin. Cholesterol was removed with the use of β-cyclodextrin while sphingomyelin was decomposed by exogenous sphingomyelinase. Cyclodextrin at 5-10 mM removed about 70% of cholesterol from the cells and abolished the assembly of FcγRII caps thereby arresting the receptors at the patching stage. Similarly, 70 mU/ml sphingomyelinase inhibited cap formation by 60%. Cholesterol and sphingomyelin depletion also suppressed the tyrosine phosphorylation of proteins which accompanied cross-linking of FcγRII. The observations indicate that cholesterol and sphingomyelin can control the interactions of tyrosine kinases with clustered FcγRII.
To downregulate expression of the β1 integrin subunit in endothelial cells, plasmid encoding the ribozyme cassette containing hammerhead ribozyme flanked at the 5' terminus by tRNAVal and at the 3' terminus by constitutive transport element sequences was constructed. When used to transfect immortalized human endothelial cell line EA.hy 926, it selectively blocked the synthesis of the β1 integrin subunit and thus inhibited migration and proliferation of the cells. Thus, this construct may be a valuable tool to control the proangiogenic phenotype of stimulated endothelial cells.
Neospora caninum is an apicomplexan parasite, which owes its importance to the fact that it represents the major infectious cause of bovine abortion worldwide. Its life cycle is comprised of three distinct stages: Tachyzoites, representing the proliferative and disease-causing stage, bradyzoites, representing a slowly replicating, tissue cyst-forming stage, and sporozoites, which represent the end product of a sexual process taking place within the intestinal tissue of the final canine host. Tachyzoites are capable of infecting a large variety of host cells in vitro and in vivo, while bradyzoites have been found mainly within the central nervous system. In order to survive, proliferate, and proceed in its life cycle, N. caninum has evolved some amazing features. First, the parasite profits immensely from its ability to interact with, and invade, a large number of host cell types. Secondly, N. caninum exploits its capability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite or vice versa). Thirdly, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long term survival of not only the parasite but also of the host cell. These three key events (host cell invasion — stage conversion — host cell modulation) represent potential targets for intervention. In order to elucidate the molecular and cellular bases of these important features of N. caninum, cell culture-based approaches and laboratory animal models are extensively exploited. In this review, we will summarize the present knowledge and achievements related to host cell and parasite cell biology.
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