Purification of antimicrobial factor from granules of channel catfish peripheral blood leucocytes
Section snippets
Materials and methods
Maintenance of channel catfish. Adult catfish were maintained in tanks at 27 °C in a recirculating water-reuse culture system at the Ecological Research Center (Dr. Bill Simco, Director), Department of Biology, University of Memphis [11], [12], [13]. Channel catfish were provided by Dr. William Wolters, USDA-ARS, Catfish Genetics Research Unit, Stoneville, MS.
Separation of leucocytes from channel catfish blood. Fifty milliliters of heparinized blood was obtained by bleeding from the caudal sinus
Results
A blood cell fraction of leucocytes was isolated from channel catfish peripheral blood by density gradient centrifugation. A procedure that specifically extracts a granular-rich sediment from human neutrophils was used for the catfish leucocytes [15]. The leucocytes were first homogenized to obtain granule-rich supernatants by low-speed centrifugation. The supernatants were then combined and subjected to high-speed centrifugation to obtain a pellet of granules. A 10% acetic acid extract of the
Discussion
We have extensively investigated immunity in the channel catfish concentrating on the complement system [5], [11], [12], [13], [28], [30], [31], [32], [33], [34], [35]. It was found that both the classical and alternative complement pathways are activated and can have a bactericidal effect against catfish pathogens. Bactericidal activity by catfish serum, mediated by the alternative complement pathway, was an important means of defense against many Gram-negative bacteria. The alternative
Acknowledgements
The authors thank the following individuals for their assistance: Dr. William Lewis, Center for Biotechnology, St. Jude Children’s Research Hospital, Memphis, TN, for doing Edman degradation for amino acid sequence analysis; Dr. Weixing Zhang, Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, for doing NMR structural analysis; Dr. Edward Umstot, Stout Mass Spectrometry Laboratory, University of Tennessee Health Science Center, Memphis, TN, for doing
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Present address: Department of Life Science and Biotechnology, Dong Eui University, Pusan, Korea.