Electronic Theses and Dissertations

Identifier

792

Date

2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biology

Concentration

Molecular Cell Science

Committee Chair

Donald D Ourth

Committee Member

King Thom Chung

Committee Member

David Freeman

Committee Member

Bill Simco

Abstract

The sea lamprey (Petromyzon marinus) belongs to the most primitive class of fish and has only innate immunity. Mannose-binding C-type lectin (MBL) was initially isolated by mannan-agarose affinity chromatography from sea lamprey plasma. The affinity-purified and 2-ME reduced lamprey MBL showed two bands of 35 kDa and 65 kDa by SDS-PAGE and Western blotting using guinea pig anti-MBL IgG as the primary antibody. N-terminal amino acid sequencing by Edman degradation for the first 10 residues gave XXXTKGCPDA. Lamprey plasma contained 261 µg of MBL/mL of plasma. Plasma protein concentration was 40.1 mg/mL. Lamprey plasma was present them in plasma at 6.5 µg MBL/mg total protein. The sea lamprey MBL specifically binds to the mannose on the surface of the pathogen Aeromonas salmonicida. Plasma concentration of lamprey lysozyme was 5 µg lysozyme/mg total protein. Lysozyme and an antifungal peptide were isolated by low molecular weight gel filtration chromatography from sea lamprey plasma. Lysozyme and antifungal activity for each fraction were determined by well diffusion assay using Gram-positive bacteria, Gram-negative bacteria and two fungal species. The molecular weight of lamprey lysozyme was 14.3 kDa. The sea lamprey lysozyme was effective against Gram-positive bacteria but not against Gram-negative bacteria or fungi. Molecular weight of the antifungal peptide was approximately 3,000 Daltons. Antifungal plasma acitivity was seen against Penicillium notatum and Aspergillus flavus. Decreased immunity from stress can lead to increased susceptibility of fish to infectious diseases. Thirty catfish were intraperitoneally injected with prednisolone acetate (PA) to stimulate a long-term stress response. Blood samples were obtained from each catfish before and for five weeks after PA injection for a total of six weeks. Sera were assayed for total protein, albumin, globulin, albumin/globulin ratios (A/G), glucose, lysozyme and MBL. Total protein concentrations decreased 50% by week six in the PA-injected catfish when compared with pre-bled sera. Lysozyme and MBL levels all increased the week following PA injection and then decreased 40-50% by week six in the stressed catfish. Catfish with the highest pre-bled total serum protein concentrations survived the longest. Chemical-induced stress affected protein synthesis by significantly decreasing both serum and innate protein concentrations.

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.

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