The structural gene for diphtheria toxin, tox, has been modified at its Sph I site by the introduction of an oligonucleotide linker encoding a unique Pst I restriction endonuclease site and a synthetic oligonucleotide encoding a-melanocyte-stimulating hormone (a-MSH). The resulting fusion gene directs the expression of a diphtheria toxin-related a-MSH hybrid protein in which the diphtheria toxin receptorbinding domain has been replaced with a-MSH sequences. The chimeric toxin has been partially purified from periplasmic extracts of recombinant Escherichia coli K-12 and has been found to be selectively toxic for a-MSH receptor-positive human malignant melanoma NEL-Mi cells in vitro.Since polypeptide fragments of microbial or plant toxins that are devoid of their cell receptor-binding domains are not cytotoxic (1, 2), it has become attractive to use these polypeptides in the assembly of hybrid toxins. A variety of cell-surface-directed agents, monoclonal antibodies (reviewed in ref.3), lectins (4, 5), and polypeptide hormones (6-8) have been chemically coupled to toxin fragments in order to direct the action of the toxin component toward specific eukaryotic cells. In general, either the A chain of the plant toxin ricin or the A fragment of diphtheria toxin has been conjugated through disulfide bond linkage to the above agents in attempts to assemble hybrid proteins that have targeted cytotoxicity. With rare exception (9, 10), those conjugates that were assembled with ricin A chain were found to be cytotoxic, whereas hybrids that were assembled with diphtheria toxin fragment A were nontoxic (11).To develop chimeric toxins that have defined structure and may be readily modified by site-directed mutagenesis, we have used solid-phase DNA synthesis and recombinant DNA methodologies for the genetic assembly of a diphtheria toxin-related a-melanocyte-stimulating hormone (a-MSH) fusion gene. The toxin-hormone chimeric gene directs the expression of a fusion protein that retains the ADPribosyltransferase (NAD+ ADP-ribosyltransferase; EC 2.4.2.30) activity and lipid-associating domains of diphtheria toxin; however, the diphtheria toxin receptor-binding domain is replaced with a-MSH sequences. The chimeric toxin has been found to be toxic for a-MSH receptor-positive human malignant melanoma (NEL-Mi) cells in culture, whereas the fusion protein is not toxic for either Chinese hamster ovary

identical in the 2 specimens indicating the absence of a selective proliferation of nonlabeled cells.In conclusion it appears that liver regeneration in contradistinction to red cell regeneration is the result of multiplication of the general liver cell population rather than multiple divisions of a small segment of that population. Summary. A study of hepatic regenerationin rats was carried out to see whether the stimulus of partial hepatectomy promotes proliferation of parenchymal liver cells or, in analogy with red cell regeneration, promotes the differentiation of stem cells to new hepatic parenchymal cells. By labeling a large proportion of hepatic cells with tritiated thymidine it was shown that the labeled cells rather than the presumably unlabeled stem cells were responsible for hepatic regeneration following partial hepatectomy.The growth of tissue cells (L cells) in suspension in a chemically defined medium, NCTC 109 with methylcellulose, has been reported by Bryant et aZ.(l). Bakken et aZ. ( 2 ) reported that this medium also supported the growth of suspension cultures of a line of human skin cells. Merchant and Hellman ( 3 ) have recently described the growth of L-M strain mouse cells in suspension culture using chemically defined 2X Eagle's medium. A serum-free medium containing lactalbumin hydrolyzate described by Higuchi (4) that supports the growth of several cell lines in monolayer cultures was modified for the growth of tissue cells in suspension culture by Tribble and Higuchi( 5 ) .This report presents a chemically defined medium formulated as a result of experiments with a cat kidney cell line, and its successful application for the growth of 5 other cell lines. Materials and methods. Preparation of the medium.Salts (except sodium bicarbonate), glucose, amino acids, and glutamine were dissolved together in hot distilled water. Vitamins, stored at -20°C as a lOOX stock solution, were added to the cooled mixture. Sodium bicarbonate and phenol red dissolved together in a small amount of water were added and the resulting solution was sterilized by filtration through a 0.2 p membrane filter and stored at 5°C as a 5X solution. Prior to inoculation, insulin, methylcellulose, streptomycin and penicillin were added aseptically and the final volume adjusted by addition of sterile distilled water. Methylcellulose was stored as a sterile 2% solution; antibiotics were stored at -20°C at lOOX concentrations. The medium used for determining amino acid requirements was prepared by adding aseptically to the filtered salts-vita,min solution individual amino acids ( 1 W X by guest on July 27, 2015 ebm.sagepub.com Downloaded from

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Three established mammalian cell lines (cat kidney, L, and HeLa cells), grown in suspension in a protein-free, chemically defined medium, were stored under liquid nitrogen in the defined medium fortified with 4 and 8 percent dimethylsulfoxide. After storage for 1 month the recovery viability was 86 percent, and cells showed normal growth upon reinoculation into the defined growth medium.

A heat‐stable chemically defined medium, free of glutamine, is described for the growth of mammalian cells in suspension culture. The presence of L‐alanine in the defined medium permitted the omission of glutamine. A 22‐fold increase in the population of a substrain of mouse L cells was obtained (3.4 × 106 cells/ml) in six days with no medium replenishment during incubation. Maximum yields (27 × 106 cells/ml) were obtained by daily medium replacement and venting of cultures. Growth was also improved in a line of cat kidney cells and HeLa cells, and in another substrain of L cells.