Author
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LOEB, MARCIA |
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JAFFE, HOWARD - NATIONAL INST. OF HEALTH |
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Submitted to: In Vitro Cellular And Developmental Biology
Publication Type: Abstract Only Publication Acceptance Date: 10/20/2000 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Two years ago, we reported that peptide factors from conditioned medium in which Manduca sexta midgut cells were grown induced isolated stem cells from Heliothis virescens midgut to differentiate in vitro to mature columnar and goblet gut cells. These peptides were identified as fragments of fetuin, one of the constituents of the bovine serum albumin that was essential for culturing these cells. We report here that two completely different peptides have been isolated from hemolymph of newly pupated Lymantria dispar. These peptides similarly induce differentiation of H. virescens midgut stem cells. Like fetuin, hemolymph was only active after treatment with chymotrypsin. Partially purified hydrolyzate was subjected to 3 successive RP-HPLC separation steps using Vydac, Zorbax and YMCODS columns. Thirty second fractions were bioassayed in groups of 5; active groups were then bioassayed singly. Well-separated peaks were subjected to Edman degradation, revealing nonapeptides EEVVKNAIA and ITPTSSLAT. No matches to known peptides were found in the BLAST database. Synthetic peptides were maximally active in vitro at 10(-6)and 10(-9) M, respectively. Lepidopteran midgut cells isolated in culture are able to maintain homeostasis or adjust proliferation and differentiation to culture conditions. Various combinations of the 4 peptides incubated with H. virescens stem cells were either no more effective than 1 peptide alone, more stimulatory, or inhibitory to differentiation, suggesting that local secretion of combinations of the peptides could regulate midgut differentiation in vivo. |
