A low molecular weight factor in lung‐conditioned medium stimulating granulocyte and monocyte colony formation in vitro

Sheridan, J. W.; Metcalf, Donald

doi:10.1002/jcp.1040810103

Cited by 164 publications

(55 citation statements)

References 34 publications

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“…In particular he showed with John Parker and John Sheridan that the CSF produced by activated lymphocytes or obtained from endotoxin-primed mouse lungs was antigenically different from M-CSF, was of a smaller size, and stimulated a greater proportion of granulocyte (G) or mixed granulocyte/macrophage (GM) colonies (11,12). Attempts to purify this GM-CSF began with John Sheridan and were continued with Jim Camakaris.…”

Section: Purification and Cloning Of The Csfsmentioning

confidence: 99%

Donald Metcalf AC. 26 February 1929 — 15 December 2014

Nicola

2016

Biogr. Mems Fell. R. Soc.

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Donald Metcalf was one of Australia's most distinguished medical researchers and is acknowledged internationally as the father of the modern field of haemopoietic growth factors. He defined the hierarchy of haemopoietic progenitor cells, purified and cloned the major molecular regulators of their growth and maturation, determined their mechanisms of action and participated in their development for clinical use in cancer patients. He received numerous awards and distinctions during his career, but was most pleased by the fact that his life's work improved human health.

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Section: Purification and Cloning Of The Csfsmentioning

confidence: 99%

Donald Metcalf AC. 26 February 1929 — 15 December 2014

Nicola

2016

Biogr. Mems Fell. R. Soc.

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“…After incubation for seven days at 37"C, the colonies were counted. Mouse lung conditioned medium prepared according to Sheridan and Metcalf [14] was used as a colony-stimulating factor. Human T-lymphocyte colony assay was performed in agar-containing glass capillaries as described by Maurer et al [15].…”

Section: As36mentioning

confidence: 99%

Isolation of a specific granulopoiesis inhibitor from calf spleen and identification as glutathione

Fetsch

Eckart

Maurer

1990

European Journal of Biochemistry

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A small peptide was isolated from calf spleen, specifically inhibiting murine granulopoiesis in vitro. Purification involved ultrafiltration, anion-exchange chromatography with a FPLC system and reversed-phase chromatography on HPLC. Determination of the amino acid composition, following acid hydrolysis and phenyl isothiocyanate and dabsyl chloride derivatization, revealed the amino acids glutamic acid, cysteine and glycine. Although the N-terminal amino group was not blocked, peptide sequencing with common techniques was not possible.Comparison of the isolated peptide with the well-known tripeptide glutathione by HPLC and fast atom bombardment (FAB)/tandem mass spectrometry showed the identity of both substances. Moreover, glutathione was found to be a specific granulopoiesis inhibitor in vitro at 10-100 nM, a so far unknown property of this well-known peptide.The term chalones was reintroduced by Bullough for physiological substances specifically inhibiting cell proliferation by a negative feedback mechanism [l]. Chalones were thought to exert a cell and tissue-specific but species-unspecific effect. Paukovits and Laerum [2] described a synthetic pentapeptide (called hemoregulatory peptide HPSb, pGluGlu-Asp-Cys-Lys) meeting these properties. Their reduced monomer is a specific in vitro and in vivo inhibitor of granulopoiesis [3], whereas the dimer stimulates granulocyte/ macrophage colony formation in vitro [4].Recently we have reported a similar specific in vitro granulopoiesis inhibitor, partially purified from calf spleen [S]. In this paper we describe the purification to homogeneity and the identification of this peptide as glutathione. MATERIALS AND METHODS Preparation of an acetone precipitateThe preparation has been described in detail by Kastner [6]. In short, calf spleen was extracted with ethanol (70%, by vol.) and centrifuged. The clear supernatant was added to acetone. The lyophilized extract was called the 'acetone precipitate'. UltrafiltrationUltrafiltration was performed in a special multichamber system (UFLA-None, Firma Schiitt, Gottingen), developed by Dr A. Kinawi of this institute. We used seven membranes with decreasing cut-off values, i.e. XM I00 A, XM 50 Abbreviations. DABITC, 4-dimethylaminoazobenzene-4'-isothiocyanate; FAB, fast atom bombardment; GSH, reduced glutathione; pGlu, pyroglutamic acid. _~was performed with a flow rate of 6 ml/h for 22 h at 4°C. The ultrafiltrates were lyophilized and stored at -20 "C. Fast protein liquid chromatographyFPLC was performed with equipment from Pharmacia, Freiburg. We used an anion-exchange column (MonoQ) with a NaCl gradient of 0-0.4 M for 20 min then 0.4-1 M for 2 min. The flow rate was 2 ml/min with a fraction size of 400 pl. Detection was at 254 nm. No buffer was used to avoid any interference with biological testing. Purijication vvith high-perjormance liquid chromatographyHPLC was performed on a Beckman system with a reversed-phase column 5 pm, Merck, Darmstadt). Solvent A was 0.1 % trifluoroacetic acid in water, solvent B was 0.1 % trifl...

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“…Mouse bone marrow and spleen cells were collected (Sheridan & Metcalf, 1973;Metcalf, 1976) immediately prior to counting and culture in semisolid agar medium.…”

Section: Cell Lines and Cell Typesmentioning

confidence: 99%

“…Unless stated to the contrary the medium contained heatinactivated FCS. In studies involving mouse bone marrow or spleen cells either a source of granulocyte-monocyte colony stimulating factor (GM-CSF) (Sheridan & Metcalf, 1973) or 2-mercaptoethanol and endotoxin were included (Metcalf, 1976).…”

Section: Clonogenic Cell Assaymentioning

confidence: 99%