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Development Of
A Cell Line From The Liver Of Atlantic Menhaden (Brevoortia tyrannus): A Model For Studies Of Bacterial Infections Mohamed Faisal1, G. Clergé2, M.H. Kothary2, M.D. Miliotis2,
D.E. Hanes2 J.W. Bier2, D. B. Shah, and B. D. Tall2,
and B. Eribo3 1Virginia
Institute of Marine Science, College of William and Mary, Gloucester Point, VA. 2JIFSAN, US
Food and Drug Administration, Washington, D.C.; 3Howard University,
Washington, D.C. The
liver is the main target organ for toxic chemicals and infectious agents in all
vertebrates, including fish. Despite continuous efforts, no permanent cell
lines of non-transformed hepatocytes have been developed. In this report we
describe a standardized single-step enzymatic digestion procedure that produced
a confluent monolayer of Atlantic menhaden liver cells. Subcultures were
successful and 144 subcultures were obtained with a doubling time of 48 hr. The
cell line, designated as AML, appears to be metabolically active as revealed by
light and electron microscopy. Over 85% of the AML cells possess one or two
highly refractile droplets that stained positively with Sudan Black B,
indicating a lipid nature. AML has 43 teleocentric chromosomes, the smallest of
which is non-paired, two submetacentric, and two metacentric chromosomes.
Western blot analysis indicated that AML cells produce high concentration of
cytochrome P-450 1-A proteins constitutively.
Because liver diseases are the
leading predisposing conditions to severe Vibrio
vulnificus infections, AML cells were chosen to investigate the mechanism
of hepatocyte invasion utilized by this pathogen. Results demonstrated that
internalization of V. vulnificus
occurred at a limited number of sites on the AML cell surface. Electron
microscopy provided evidence that internalization took place at the apical and
peripheral cell surface at 5-7 foci of infection (2-5 bacteria/focus). Results also indicated that V. vulnificus invasion was dependent on both microtubulin and
microfilament polymerization/ depolymerization. Activation of both s-protein
tyrosine kinase (sPTK) and protein kinase C (PKC) took place
during the invasion process. Our data
also demonstrated that V. vulnificus uptake
and bacterial-mediated cytotoxicity of AML cells are independent events.
Together these data suggest that the AML cells are as alternative study to
study hepatic processes.
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