Agents facilitating the electric field-induced fusion of intact rabbit erythrocytes.

In the recent years, the role of specific membrane active agents in the electrofusion process has started to draw attention, and it has been found that the presence of various substances in the cell medium can affect the fusion process either in a positive or negative way. In this work, the effect of several proteins, bivalent cations and antibiotics was tested with respect to their ability to protect intact erythrocytes from hemolysis and facilitate the fusion process. The effect of different sugars was also studied. Among the different proteins, pronase and proteinase were found to be the most effective. With respect to bivalent cations, Ca2+ and Mn2+ were more effective while Mg2+ was less important. From the antibiotics, penicillin caused a negative effect while streptomycin acted positively. Finally, glucose medium was found to be the most effective compared to all sugars tested. The results indicated that there are strong differentiations of the induced effects caused by each substance, and some possible mechanisms of action of these agents on the erythrocyte membrane were discussed.

Loading of intact rabbit erythrocytes with fluorophores and the enzyme pronase by means of electroporation.

The application of electric field pulses in cell suspensions is known to alter membrane integrity, resulting in increased membrane permeabilization. This field-induced membrane poration provides the means to load cells with a variety of external substances, useful for clinical applications. In this work, intact rabbit erythrocytes were successfully loaded with low molecular weight fluorescent probes and with the high molecular weight enzyme pronase, which has been shown to mimic the effects of insulin. Attachment of the enzyme onto the cell surface was also achieved by modifying the applied pulse parameters. Both applications were efficient and accompanied by high cell survival rates. In this way, biological carriers loaded with active substances were produced, offering the potentials for useful clinical applications, either for diagnostic or therapeutic purposes.

Sequence analysis and expression of the host-protective immunogen VP2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type I strains.

The host-protective antigen VP2 of a variant strain of infectious bursal disease virus (IBDV) which emerged from a vaccinated flock and is able to circumvent vaccination with classic type I strains of IBDV, was cloned and its nucleotide sequence determined. Virus-neutralizing monoclonal antibodies (MAbs) raised against the Australian 002-73 strain of IBDV did not react or reacted only very weakly with the expression product of the variant virus. The deduced amino acid sequence of VP2 from the variant strain differed in 17 residues from that of the Australian strain and in eight positions from a consensus sequence compiled from six type I strains of IBDV. All the amino acid changes mapped within the central, variable region of VP2, which forms the conformational epitope recognized by virus-neutralizing MAbs. Changes in the two hydrophilic regions at either end of this fragment were unique to the variant virus and were crucial for its ability to escape the virus-neutralizing antibodies induced by vaccination with a standard type I vaccine.

Purification and characterization of two wheat-embryo protein phosphatases.

Two protein phosphatases (enzymes I and II) were extensively purified from wheat embryo by a procedure involving chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, DEAE-Sephacel and Ultrogel AcA 44. Preparations of enzyme I (Mr 197,000) are heterogeneous. Preparations of enzyme II (Mr 35,000) contain only one major polypeptide (Mr 17,500), which exactly co-purifies with protein phosphatase II on gel filtration and is not present in preparations of enzyme I. However, this major polypeptide has been identified as calmodulin. Calmodulin and protein phosphatase II can be separated by further chromatography on phenyl-Sepharose CL-4B. Protein phosphatases I and II do not require Mg2+ or Ca2+ for activity. Both enzymes catalyse the dephosphorylation of phosphohistone H1 (phosphorylated by wheat-germ Ca2+-dependent protein kinase) and of phosphocasein (phosphorylated by wheat-germ Ca2+-independent casein kinase), but neither enzyme dephosphorylates a range of non-protein phosphomonoesters tested. Both enzymes are inhibited by Zn2+, Hg2+, vanadate, molybdate, F-, pyrophosphate and ATP.