An attempt was made to isolate the cell proliferation stimulation factors in the supernatant of embryo carcases and adult muscles of chickens. Evidence was obtained for the presence of at least two or more stimulating factors in both the embryonic and adult muscular supernatants. These factors did not require a supplement of sera or other supporting agents. Furthermore, the use of the salting-out method with ammonium sulfate revealed two or more growth stimulants in the supernatant of chick cells.</p

Hatase, Osamu

Itano, Toshifumi

Kuramitsu, Makoto

Nisida, Isamu

Takahashi, Fumio

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Plural growth factors in the supernatant of embryos and adult muscles of chickens

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 31, Issue 1 1977 Article 8FEBRUARY 1977Plural growth factors in the supernatant ofembryos and adult muscles of chickensFumio Takahashi∗ Osamu Hatase† Makoto Kuramitsu‡Toshifumi Itano∗∗ Isamu Nisida††∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,††Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Plural growth factors in the supernatant ofembryos and adult muscles of chickens∗Fumio Takahashi, Osamu Hatase, Makoto Kuramitsu, Toshifumi Itano, andIsamu NisidaAbstractAn attempt was made to isolate the cell proliferation stimulation factors in the supernatantof embryo carcases and adult muscles of chickens. Evidence was obtained for the presence ofat least two or more stimulating factors in both the embryonic and adult muscular supernatants.These factors did not require a supplement of sera or other supporting agents. Furthermore, theuse of the salting-out method with ammonium sulfate revealed two or more growth stimulants inthe supernatant of chick cells.∗PMID: 140593 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 31,91-94 (1977)BRIEF NOTEPLURAL GROWTH FACTORS IN THE SUPERNATANTOF EMBRYOS AND ADULT MUSCLESOF CHICKENSFumio TAKAHASHI, Osamu HATASE, Makoto KURAMITSU,Toshifumi hANG and Isamu NISIDADepartment of Physiology, Okayama UniversityMedical School, Okayama 700, JapanReceived December 1, 1976Abstract. An attempt was made to isolate the cell proliferationstimulating factors in the supernatant of embryo carcasses and adultmuscles of chickens. Evidence was obtained for the presence of atleast two or more stimulating factors in both the embryonic and adultmuscular supernatants. These factors did not require a supplementof sera or other supporting agents. Furthermore, the use of thesalting-out method with ammonium sulfate revealed two or moregrowth stimulants in the supernatant of chick cells.Many investigators have attempted to purify the active components fromchick embryo extracts that promote cell proliferation but the successful isolationand purification of the stimulating factors as protein have not been reported (1,2,3). We have previously investigat~d (4) the reasons for the difficulty in puri-fying the cellular stimulants and have tried to purify such active componentsfrom the supernatants of embryo carcasses a~d adult muscles of chickens usingvarious preparation methods. Under ethanol fractionation, two or more cellproliferation stimulating factors were tentatively found in the supernatant ofembryo carcaSSES and adult muscles (4). The changes in stimulating activities andphysicochemical properties were remarkable during embryological development,but the stimulating activities were not fractionated. To further elucidate wheth-er there are two or more stimulants in the supernatant under ethanol fractiona-tion and to determine whether ethanol fractionation is unsuitable for purificationof such active components, we used the salting-out method with ammoniumsulfate. In the present paper, we report on the findings of this probe. The resultsindicated that there were at least two or more cellular proliferation stimulatingfactors in the supernatant of embryo carcasses and adult muscles of chickens.The supernatant (S2) of chick embryo (Em) carcasses (IO-day-old) and adultchicken muscles (M) were prepared by the procedures described in a previous911Takahashi et al.: Plural growth factors in the supernatant of embryos and adultProduced by The Berkeley Electronic Press, 197792 F. TAKAHASHI, O. HATASE, M. KURAM1TSU, T.1TANO and 1. N1SIDApaper (4). The S2 was slowly saturated by addition of solid ammonium sulfateup to 30% and 60% in the presence of 0.5 mM 2-mercaptoethanol, and the pre-cipitates were collected stepwise by centrifugation and were lyophilized afterextensive dialysis against 0.5 mM 2-mercaptoethanol and I mM Tris-HOI, pH7.4. The stimulating effects of these fractions were assayed by secondary cul-tured chick embryo cells (OEF) as described in the previous papers (4,5).Table I shows that the Em supernatant(EmS2) contained stimulating factor~s)of OEF proliferation and that the active components were independently pre-cipitated in 30% fraction (Em30), 60% fraction (Em60) and unprecipitated frac-tion (EmSup) by salting-out with ammonium sulfate. The relative stimulatingactivities in each fraction were similar to each other. The muscular fractionsalso contained stimulants in all three fractions (Table 2). These results stronglyTABLE 1. STIMULATING EFFECTS OF EMBRYONIC FRACTIONS ON CHICKEMBRYO CELL (CEF) PROLIFERATIONSystemsResting control (RT)RT + EmSzcRT + Em30cRT + Em60cRT + EmSupcCell number X l04 a4.907.758.048. 788.54Stimulation percentbo+ 58+ 64+ 79+ 74a CEF in resting media (RT) composed of MEM + 2% TPB were incubated for 48 hours at37°C in a C02 gas incubator with or without sample fractions (50 fig/ml). The cells werecounted by a Coulter counter after trypsinization. Triplicate plastic dishes were used forone assay system and the standard errors were within 7.8 to 9.6% of the mean value (M).b Stimulation percent was calculated by : [Cell number in experimental system-Cell numberin RT] -0-- Cell number in RTx 100.c EmSz was the original supernatant of Em; Em30 was fractionated with 30% saturation ofammonium sulfate; Em60 was 60% saturation fraction; and EmSup was unprecipitatedfraction after 60% saturation of ammonium sulfate.TABLE 2. STIMULATING EFFECTS OF MUSCULAR FRACTIONS ON CEF PROLIFERATIONSystemsResting control (RT)RT + MSzcRT + M30cRT + M60cRT + MSupcCell number X 104a6·69.69.69.08.4Stimulation percentUo+ 45+ 45+ 36+ 27a Same as footnote a in the legend of Table 1.b Same as footnote b in the legend of Table l.c MS2 was the supernatant fraction of M; M30 was 30% precipitated fraction; M60 was 60%precipitated fraction; and MSup was unprecipitated fraction after 60% saturation of am-monium sulfate.2Acta Medica Okayama, Vol. 31 [1977], Iss. 1, Art. 8http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss1/8Plural Growth Factors in Chick Cells 93support the possibility that there are two or more stimulants in the supernatant,and these results coincide with those obtained by ethanol fractionation. Bothmethods of fractionation revealed the presence of plural stimulating factors inthe supernatant. Isoelectric precipitation also showed two stimulants in thesame supernatant (data not shown).Fig. I shows the electrophoretic pattern of Em30, Em60, M30 and M60 in7.5% polyacrylamide gel, and common components are found in each fraction.(-) T T T0.03 0.15 Q25jfl ';:/ ';, fI. \ '1 (+)0.95Fig. 1. Densitometrical pattern of embryonic and muscularfractions in polyacrylamide gel electrophoresis.Polyacrylamide gel (7.5%) electrophoresis was performed by themethod of Davis (7) in Tris-glycine buffer, pH 8.5. The samples(100 fig) were charged and electrophoresed for 150 min at roomtemperature. M30, -.-; M60, -; Em30, ; Em60, ----The Rfs were 0.03,0.15, 0.25, 0.58 and 0.95. The active components in thebanded patterns are possible subjects of future investigations on stimulating ac-tivity.These stimulating factors may be different from the fibroblast growth factor(FGF) reported by Gospodarowicz (6), because FGF required supplements ofhydrocortisone and insulin to stimulate cell growth in absence of sera, but ourfactors stimulated growth without any supplement.lt is concluded that there are at least two stimulating factors of CEF proli-feration in the supernatant of embryo carcasses and adult muscles of chickens.REFERENCES1. Baker, E. and Carrel, A.: Action on fibroblasts of the protein fraction of embryonictissue extract. ]. Exp. Med. 44, 387-395, 1926.2. Rosenberg, S., Zitcer, E. and Kirk, P. L.: Tissue culture studies; Isolation of activecomponents in the ultrafilterable portion of the chick embryo extract. ]. Gen. Physiol.37, 231-238, 1953.3. Coon, H. G. and Cahn, R. D.: Differentiation in vitro. Effects of sephadex fractions of3Takahashi et al.: Plural growth factors in the supernatant of embryos and adultProduced by The Berkeley Electronic Press, 197794 F. TAKAHASHI, O. HATASE, M. KURAMlTSU, T. ITANO and 1. NISIDAchick embryo extract. Science 153, 1116-1119, 1966.4. Hatase, 0., Takahashi, F., Fujii, T., Hidaka, M. and Nisida, I.: Cell proliferation stimu-lating factors in the supernatant of chick embryos and adult muscles of chickens of thesame strain. Acta Med. Okayama 30, (6), 397-402, 1976.5. Vogt, P. K.: Focus assay of Rous sarcoma virus. In Fundamental Techniques in ViToloO,ed. K. Habel and N.P. Salzman, Academic press, New York, 198-211, 1969.6. Gospodarowicz, D.: Purification of a fibroblast growth factor from bovine pituitary. j.Biol. Chern. 250, 2515-2520, 1975.7. Davis, B. J.: Disc electrophoresis II. Method and application to human serum proteins.Ann. N. Y. Acad. Sci. 121, 404-427, 1964.4Acta Medica Okayama, Vol. 31 [1977], Iss. 1, Art. 8http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss1/8

Plural growth factors in the supernatant of embryos and adult muscles of chickens

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