of incorporatcd tritium lahellecl thymidine is distributed to thc daughter (.ell at mitosis( 1 ) one can exlmt that 1 y atlministering tritiutn labelled thymidine at 6 hour intervals, virtually all cells dividing during the period between booster injection of antigen and sacrifice would be exposed to, and incorporate labelled thymidine. The value of 92% stated as the labelling index of the plasma cell series here is probably conservative and may actually approach 100%. This low value might result )from the initiation and coinpletion of DNA synthesis in some cells between esposures to isotope or from technical limitations in the sensitization of the radiographic emulsion by the label in some cells. These factors, alone or in combination, would tend to lower the observed incidence af labelling. The results presented are in general agreement with those of other investigators employing similar technics (8,10,11) who have implicated a rapidly proliferating population of immature lymphablasts and plasmablasts as the chief source of plasma cells arising after antigenic stimulation. As a corollary. the large percentage of labelled antibody containing cells found in these experiments indicate that most plasma cells do not arise by direct, non-mitotic differentiation from small lymphocytes. The present studies could not offer quantitative data concerning the relative mitotic activity of the various stages leading to development of the mature plasma cell. However, by the use of colchicine and by short in vitro incubation of cells with thymidine it has been shown that functionally differentiated antibody producing cells can divide, indicating that mitotic activity within the family of antibody producing cells is not a n exclusive property of the immature or pre-CII rsor f orins.Suiiiiiinry. 14 technical approach for the study of the kinetics of antibody producing cells is possible by combination of autoradiography and the fluorescent antibody technic. Results show that: 1. Almost all antibody containing cells present by day 4 of a secondary response are newly formed cells arising ifrom mitotic division of a precursor sometime after antigenic stimulation; 2. Most plasma cells do not arise by direct, non mitotic differentiation from lymphocytes; and 3. Functionally differentiated antibody containing cells can divide.
The in vitro synthesis of collagen has been demonstrated in sponge biopsy connective tissue ( 1 ) , carrageenin granulomata ( 2 ) , rat uterus(3), rat cartilage(4) and rat bone(5) by either conversion of C14-lysine to C14-hydroxylysine or C14-proline to C14-hydroxyproline. Recently, homogenates from chick embryo have been reported capable of converting C14-proline to C14-hydroxyproline (6). Ascorbic acid was reported to be essential in the hydroxylation of proline to hydroxyproline in carrageenin granulomata from guinea pig ( 7,8). Designation of the hydrogen atoms of proline involved in the hydroxylation has been attempted by two groups of workers (8,9), each using both C14-and tritiated-proline; their results are not in agreemen t . Prockop et aZ.(lO), Scharpenseel et aZ.(1 1), and Fujimoto et aZ.( 12), using Ozs as a tracer, have reported that atmospheric oxygen rather than the oxygen of water is required for synthesis of collagen. The metabolism of collagen is still obscure. This study deals with a systematic survey of the op'timal conditions for collagen synthesis in sponge biopsy connective tissue.Methods. Sponge biopsy connective tissue, 2 weeks old, was obtained by the method ofBoucek and Noble( 13). Tissue slices ( 1 .OO g) were incubated in 5 ml Krebs-Henseleit medium( 14) with 1 ml saline containing 1 pc uniformly labelled L-pr01ine-C~~ (Schwarz BioResearch, Inc., specific activity 1.09 pc/ mg or 800 @c/mg) at 37OC, pH 7.35-7.45 under an atmosphere of 95% 02-5% COa, unless otherwise stated. After 6 hours of incubation, the slices were removed, blotted and homogenized in 0.85% sodium chloride containing 0.25% L-proline. The mixture was *This work was supported in part by U.S.P.H.S. grant.centrifuged at 15,000 x g for one-half hour and decanted. The residue was extracted with 0.1 N NaOH overnight and centrifuged at 105,OOO X g for 1 hour. This extraction was repeated once for 2 hours. To avoid the contamination of noncollagenous protein and C14-proline in the incubation medium, the NaCl solution and the base were discarded. Collagen was extracted from base-soluble residue by Fitch's method( 15). The purity of the collagen was indicated by demonstrating an hydroxyproline content of 13%. The collagen samples were hydrolyzed in 6N HCl at 110' for 16 hours in sealed tubes. Aliquots were taken for N2 determination by Kjeldahl digestion and Conway diffusion techniques (16). Radioactivity was counted in a low background (2 counts per minute) gas flow counter with micromil window. Our counting efficiency was 40%. Data were not corrected for counting efficiency. Specific activity was expressed as cpm/mg protein. Hydroxyproline and proline were separated on Whatman #4 paper in 2 solvent systems, 77% ethanol or water saturated phenol in a vapor phase of NHS (0.3% NH4OH) and HCN ( 5 % NaCN). Both solvents were proven to be satisfactory for separation of hydroxyproline from proline. Glutamic acid synthesis was negligible during 6 hours of incubation. The amino acids were eluted from the paper with water. Aliquots wer...
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