- Email: [email protected]
Low molecular weight RNA in lymphocytes of chronic lymphocytic leukemia
536
Preliminary
notes
filter. Incubation of red cells with supernatants from macrophages cultivated overnight gave no chromium release. L cells cultivated under similar conditions did not show any hemolytic activity and phase contrast micrograph from L cell cultures did not show damaged red blood cells or ghosts attached. Cultivating macrophages in a nonconfluent layer gave much less isotope release compared with the usual confluent layer.
The efficiency of the cytotoxic effect of macrophages should be noted. In our experiment IO6 cells were able to cause complete chromium release from IO7 red blood cells at 5 h. This is an efficiency of at least 200 times that exerted by the lymphocytes on target cells [6]. It is thus clear that even small admixtures of macrophages to lymphocyte suspension will greatly influence experimental results under conditions where macrophages are cytotoxic. A more complete account of our results is under preparation.
Discussion This study demonstrates that macrophages may exert strong cytotoxic activity. That this effect is extracellular and not mediated by phagocytosis and subsequentlysosomal breakdown of the target cells, is demonstrated by the release of intact hemoglobin molecules [3]. The extracellular position of damaged red cells was also directly demonstrated with phase contrast microscopy. Our experiments indicate that the cytotoxic action is dependent on close contact with the target cells. It appears that some kind of activation is necessary to render the macrophages cytotoxic. This is in keeping with recent observations made by Evans & Alexander [2]. However, in our system it has not proved essential to immunize the animals before collecting macrophages with cytotoxic potential. On the contrary, the phenomenon is fully operative in a syngenic situation, provided that the macrophages have been ‘turned on’ by contact with heparin or by cultivation on glass surfaces for some hours. Preliminary experiments in our laboratory have demonstrated that activation may also be brought about by the presenceof concanavalin A and Zn2+. The degree of cytotoxicity seemsto be dependent on the serum present-may be as an effect of conglutinins or similar substances causing close contact between target cells and macrophages. Exptl Cell Res 75 (1972)
References I. Cohn, 2 A &Benson. B. J exutl med 21 (1965) 153. 2. Evans, R & Alexander, P, Nature new bibI 236 (1972) 168. 3. Ehrenreich, B A & Cohn, 2 A, J cell biol 38 (1968) 244. 4. Granger, G A & Weiser, R S, Science 145 (1964) 1429. 5. Flatmark, T & Sobstad, G, Inst of biochemistry, University of Bergen, Norway. 6. Holm, G, Perlmann, P & Werner, B, Nature 203 (1964) 841. 7. M@ller, G, Transpl proc 3 (1971) 15. 8. Perlmann, P, Perlmann, H & Holm, G, Science 160 (1968) 306. Received July 25, 1972 Revised version received September 6, 1972
Low molecular weight RNA in lymphocytes of chronic lymphocytic leukemia R. W. BILLINGTON and RUTH F. ITZHAKI, Paterson Laboratories, Christie Hospital and Halt Radium Institute, Withington, Manchester M20 9BX, UK
RNA was extracted from lymphocytes of patients with chronic lymphocytic leukaemia, and analysed by polyacrylamide gel electrophoresis. The main difference between the leukaemic cells and normal lymphocytes was the large amount of low molecular weight (4-10s) RNA found in the leukaemic cells. This RNA was found to be a nuclear fraction, and is probably similar to SnRNA of other cell types.
The lymphocytes of patients with chronic lymphocytic leukaemia (CLL) are broadly similar morphologically to those found in
Preliminary notes normal individuals. However they exhibit certain functional defects which may involve abnormal RNA metabolism [l]. We report here some preliminary studies on the composition of the RNA in CLL lymphocytes; these revealed unusually large amounts of low molecular weight components. Low molecular weight nuclear RNA (SnRNA) has been reported in various tissues, e.g. normal lymphocytes [2, 31, HeLa cells [4, 51, Novikoff hepatoma [6-81 and Yoshida and Ehrlich ascites tumour cells [9-l 11.
531
t-
Materials and Methods CLL lymphocytes were prepared from fresh heparinised blood taken from patients attending Manchester Royal Infirmary or this hospital. The blood was layered on to an equal volume of solution containing 24 parts of 90 % Ficoll and 10 parts of 34 % Triosil (Glaxo) and centrifuged at 400 g for 20 min at 18°C [12]. The lymphocytes separated as a single band, and could be easily removed by Pasteur pipette. Normal lymphocytes were prepared by Chalmers’ defibrination technique [13], from the peripheral blood of volunteers, because of the greater possibility of platelet contamination. RNA was extracted either immediately after nrenaration. or, if necessary. after storage at -2O’C by the phenol method, basically as described by Kay & Cooper for lymphocytes 1141.‘Cold extracts were- carried- out at 0°C without -sodium lauryl sulphate (SLS) whereas the 40” and 60°C extracts were carried out in the presence of 0.5 % SLS. In each case the second phenol extraction was carried out at 0°C. After extraction, the RNA was treated with ribonuclease-free DNAase (Worthington) for 15 min at 0°C and reprecipitated with 2 vol of ethanol. RNA was separated into its components by polyacrylamide gel electrophoresis as described by Loening [15]. Differential centrifugation to fractionate the subcellular components was carried out under two different regimes in an effort to locate the cellular position of the RNA species in question. (1) This method was devised primarily for the preparation of ribosomes 1161.as follows: lvmnhocvtes were allowed to swell for-i0 min in medium LS; containing 10 mM NaCI, 20 mM Tris-HCI buffer (nH 7.5) and 1.4 mM MaCI,. They were then disrupted-by 15 strokes of the plugg& in a Dounce homogeniser with a clearance of 0.002 inch. The homogenate was centrifuged at 6 000 g for 6 min and the pellet retained, being referred to as ‘nuclear fraction’. The supematant was spun at 120 000 g for 1 h, giving the ribosomal pellet, together with a ‘supernatant’ fraction. (2) The second method used was designed to produce purified nuclei [17]. The lymphocytes were susnended in medium LS as described above and then broken by 100 to 200 strokes of the pestle in the tight-fitting Dounce homogeniser. The homogenate was centrifuged at 850 g for
L
+
Fig. I. Abscissa: mobility in gel; ordinate:
Az6,.
Scans of polyacrylamide gel electrophoresis of total cellular RNA. (A) Normal lymphocytes; (B) Lymphocytes from patients with chronic lymphocytic leukaemia. Low molecular weight peaks are labelled a to e. Peak a contains 5S ribosomal RNA. Gels were of 5 % acrylamide, run for 2 h at 5 mA/gel. 10 min, to give a crude nuclear pellet, which was purified by the method of Blobel & Potter [18]. The supematant was centrifuged at 12000 g for 20 min, to yield a ‘mitochondrial’ fraction, and subsequently at 120 000 g to yield ribosomal and supernatant fractions.
Results and Discussion Electrophoresis of RNA from CLL lymphocytes on 50 % polyacrylamide gels revealed a series of peaks of optical density in the 4-10s region of the gel. The peaks were present also in RNA from normal lymphocytes, but in much smaller amounts (fig. 1). These low molecular weight RNA species had similar electrophoretic mobilities to SnRNA reported in other cell types [2-111. However in CLL cells the peaks represented about 4% of the total RNA, considerably more than the 0.5 % or less reported elsewhere [2-II]. The low molecular weight peaks could be extracted from intact cells by O”, 40” or 60°C phenol extractions, with no apparent diffference in Exptl Cell Res 75 (1972)
538
Preliminary notes
efficiency. The peaks were found in all sixteen CLL patients studied, regardless of leucocyte count (10 000-250 000 cells/mm3) or stage of the disease. 2.4 % gels revealed no differences in ribosomal or transfer RNA between normal or CLL lymphocytes. To ensure that these large amounts of small molecular weight RNA did not represent an artefact of extraction, the following tests were carried out. (1) RNA was extracted both from freshly prepared and also from previously frozen lymphocytes, but in both cases the 4-10s region of the gel was identical; (2) lymphocytes were prepared from peripheral blood by the defibrination technique of Chalmers [12], as well as by the Ficoll/Triosil gradient method to ensure that the different techniques did not account for the differences between normal and leukaemic cells. In fact, the RNA appeared to be identical whichever cell separation technique was used. (3) Omission of the DNAase treatment during the extraction procedure did not affect the appearance of the low molecular weight RNA peaks [4]. The peaks were removed by RNAase treatment. (5) Omission of precautions against ribonuclease action during extraction, i.e. control of temperature, absence of bentonite from extraction medium, did not change the amount of material in low molecular weight peaks, suggesting that the material is not an artefact produced by breakdown of larger RNA molecules during extraction. These results indicate that these peaks are genuine RNA species and, in such quantities, are a characteristic of chronic lymphocytic leukaemia. To establish the cellular localisation of the peaks, the lymphocytes were fractionated by differential centrifugation. Since the RNA could be extracted at 0°C without SLS even from fresh tissue, it was thought that this indicated a cytoplasmic location [14] possibly as a ribosomal compoExptl Cell Res 75 (1972)
nent. Accordingly, a ribosomal pellet was prepared, and the RNA was extracted from this and from the other fractions produced during the preparation. Analysis of the RNA from each fraction on 5 % gels shows 5S RNA in the ribosomal fraction, but none of the other small molecular weight components. In fact, the fraction that spun down at 6 000 g contained the 4-10s components. This fraction probably consists mainly of nuclei. In an effort to confirm that these low molecular weight components are of nuclear origin, nuclei were prepared and purified as described. The nuclei obtained were divided into 2 batches, the first batch being extracted at 0°C without SLS, and the second at 60°C with 0.5% SLS. Both methods extracted the low molecular weight RNA components from the nuclei. Preparations of RNA from other cellular fractions, i.e. 12 000 g and 120 000 g pellets and 120 000 g supernatant show no signs of these components. Thus it appears that the low molecular weight RNA speciesfound in leukaemic lymphocytes are similar to the SnRNA previously reported from various tissues. Whether the presence of increased amounts of these nuclear components is related in any way to the inactive nature of the lymphocytes in CLL remains to be seen. It is hoped to investigate the kinetics of labelling of this fraction in both normal and CLL cells in an effort to further characterise it, and possibly to assign it a role in cell function. We wish to thank the Medical Staff of the Clinical Haematology Department, Manchester Royal Infirmary. and of the Radiotherapy and Pathology departm&ts of the Christie Hospital for their cooperation. The work was supported by a grant from the Leukaemia Research Fund.
References 1. Havemann, K & Rubin, A D, Proc sot exptl biol med 127 (1968) 668.
Preliminary notes 2. Hellung-Larsen, P, Tyrsted, G & Frederiksen, S, 3. 4. 5. 6. 7.
Abstract VII meeting of Nordic Society for Cell Research. Exptl cell res 67 (1971) 10. Howard, E & Stubblefield, E, Exptl cell res 70 (1972) 460. Weinberg, R, Biochim biophys acta 190 (1969) 10. Rein, A & Penman, S, Biochim biophys acta 190 (1969) 1. Busch, H & Smetana, K, The nucleolus (ed H Busch & K Smetana) pp. 285-317. Academic Press, New York and London (1970). Hodnett, J L & Busch, H, J biol them 243 (1968)
6334. 8. Prestayko, A W, Tonata, N & Busch, H, J mol
bio147 (1970) 505. 9. Prestayko, A W, Tonata, N, Carol-Lewis, B &
Busch, H, J biol them 246 (1971) 182. IO. Frederiksen, S, Tnnnesen, T & Hellung-Larsen,
P, Arch biochem biophys 142 (1971) 227. 11. Hellung-Larsen, P & Frederiksen, S, Anal biothem 40 (1971) 227. Harris, H & Ukaejiofo, E 0, Lancet 2 (1969) 237. ::: Coulson, A S & Chalmers, D G, Immunology 12 (1967) 417. 14. Cooper, H L & Kay, J E, Biochim biophys acta 147 (1971) 322. 15. Loening, U E, Biochem j 102 (1967) 251. 16. Kay, J E, Ahern, T & Atkins, N, Biochim biophys acta 147 (1971) 322. 17. Laszlo, J, Ta-Fu Huang, A & Kremer, W B, Methods in cancer research (ed H Busch) vol. 5, p. 377. Academic Press, New York and London (1970). 18. Blobell, G & Potter, V R, Science 154 (1966) 1662. Received August 8, 1972
Some characteristics of the morphogenic inhibitor in blastocoelic fluid from sea urchin embryo of Zoology, University of California, Berkeley, Calif. 94720 and Bodega Marine Laboratory, University of California, Bodega Bay, Calif 94923 USA W. E. BERG, Department
External application of the blastocoelic fluid from the sea urchin embryo was found to inhibit gastrulation reversibly [l]. The following are additional observations on the characteristics of the inhibitor. Blastocoelic fluid (hereafter referred to as BF) preparations were obtained, as previously described, by removal of the hyaline layer at the early gastrula stage and flattening the embryos by centrifugal force. The embryos
539
pack down and the clear viscous supernatant is presumed to be, in large part, fluid forced out from the blastocoel through the ruptured gastrular walls. An approximate assayfor the potencies of the preparations consisted of serial dilutions in microliter culture chambers. Test embryos for determining the degree of inhibition of morphogenesis were added at a late blastula stage. The original study was confined to embryos of Lytechinus pictus. Cross tests of BF solutions have now been carried out between the echinoderms L. pictus, Strongylocentrotus purpuratus, and Dendraster excentricus. No species specificity of BF from the 3 echinoderms was observed, although there was noticeable variability of the inhibitory potencies and the sensitivity of the embryos. In general, gastrulation in S. purpuratus embryos was more sensitive to the inhibitor and morphogenesis was blocked in relatively high dilutions of the various BF preparations. BF from Dendraster embryos was considerably weaker than from the other species; usually gastrulation of test embryos was very slowly completed leading to radially symmetrical postgastrulae. BF from L. pictus embryos had no specific morphogenic effect on gastrulation of Urechis caupo embryos whereas it had strong effects on development of embryos of Mytilus edulis and Ciona intestinalis. For example, M. edulis embryos at hatching exposed to a l/2 dilution of L. pictus BF generally disintegrated. In l/4 and l/S dilutions they remained motile and developed an apical tuft, after 48 h a velum formed but no shell and little sign of internal organization. Due to the opaqueness of the embryos, it was not possible to observe whether this inhibition of morphogenesis and differentiation stemmed from an initial effect on gastrulation. L. pictus BF also inhibited development of C. intestinalis embryos. In l/2 dilutions the embryos, added shortly before Exptl Cell Res 75 (1972)
Preliminary
notes
filter. Incubation of red cells with supernatants from macrophages cultivated overnight gave no chromium release. L cells cultivated under similar conditions did not show any hemolytic activity and phase contrast micrograph from L cell cultures did not show damaged red blood cells or ghosts attached. Cultivating macrophages in a nonconfluent layer gave much less isotope release compared with the usual confluent layer.
The efficiency of the cytotoxic effect of macrophages should be noted. In our experiment IO6 cells were able to cause complete chromium release from IO7 red blood cells at 5 h. This is an efficiency of at least 200 times that exerted by the lymphocytes on target cells [6]. It is thus clear that even small admixtures of macrophages to lymphocyte suspension will greatly influence experimental results under conditions where macrophages are cytotoxic. A more complete account of our results is under preparation.
Discussion This study demonstrates that macrophages may exert strong cytotoxic activity. That this effect is extracellular and not mediated by phagocytosis and subsequentlysosomal breakdown of the target cells, is demonstrated by the release of intact hemoglobin molecules [3]. The extracellular position of damaged red cells was also directly demonstrated with phase contrast microscopy. Our experiments indicate that the cytotoxic action is dependent on close contact with the target cells. It appears that some kind of activation is necessary to render the macrophages cytotoxic. This is in keeping with recent observations made by Evans & Alexander [2]. However, in our system it has not proved essential to immunize the animals before collecting macrophages with cytotoxic potential. On the contrary, the phenomenon is fully operative in a syngenic situation, provided that the macrophages have been ‘turned on’ by contact with heparin or by cultivation on glass surfaces for some hours. Preliminary experiments in our laboratory have demonstrated that activation may also be brought about by the presenceof concanavalin A and Zn2+. The degree of cytotoxicity seemsto be dependent on the serum present-may be as an effect of conglutinins or similar substances causing close contact between target cells and macrophages. Exptl Cell Res 75 (1972)
References I. Cohn, 2 A &Benson. B. J exutl med 21 (1965) 153. 2. Evans, R & Alexander, P, Nature new bibI 236 (1972) 168. 3. Ehrenreich, B A & Cohn, 2 A, J cell biol 38 (1968) 244. 4. Granger, G A & Weiser, R S, Science 145 (1964) 1429. 5. Flatmark, T & Sobstad, G, Inst of biochemistry, University of Bergen, Norway. 6. Holm, G, Perlmann, P & Werner, B, Nature 203 (1964) 841. 7. M@ller, G, Transpl proc 3 (1971) 15. 8. Perlmann, P, Perlmann, H & Holm, G, Science 160 (1968) 306. Received July 25, 1972 Revised version received September 6, 1972
Low molecular weight RNA in lymphocytes of chronic lymphocytic leukemia R. W. BILLINGTON and RUTH F. ITZHAKI, Paterson Laboratories, Christie Hospital and Halt Radium Institute, Withington, Manchester M20 9BX, UK
RNA was extracted from lymphocytes of patients with chronic lymphocytic leukaemia, and analysed by polyacrylamide gel electrophoresis. The main difference between the leukaemic cells and normal lymphocytes was the large amount of low molecular weight (4-10s) RNA found in the leukaemic cells. This RNA was found to be a nuclear fraction, and is probably similar to SnRNA of other cell types.
The lymphocytes of patients with chronic lymphocytic leukaemia (CLL) are broadly similar morphologically to those found in
Preliminary notes normal individuals. However they exhibit certain functional defects which may involve abnormal RNA metabolism [l]. We report here some preliminary studies on the composition of the RNA in CLL lymphocytes; these revealed unusually large amounts of low molecular weight components. Low molecular weight nuclear RNA (SnRNA) has been reported in various tissues, e.g. normal lymphocytes [2, 31, HeLa cells [4, 51, Novikoff hepatoma [6-81 and Yoshida and Ehrlich ascites tumour cells [9-l 11.
531
t-
Materials and Methods CLL lymphocytes were prepared from fresh heparinised blood taken from patients attending Manchester Royal Infirmary or this hospital. The blood was layered on to an equal volume of solution containing 24 parts of 90 % Ficoll and 10 parts of 34 % Triosil (Glaxo) and centrifuged at 400 g for 20 min at 18°C [12]. The lymphocytes separated as a single band, and could be easily removed by Pasteur pipette. Normal lymphocytes were prepared by Chalmers’ defibrination technique [13], from the peripheral blood of volunteers, because of the greater possibility of platelet contamination. RNA was extracted either immediately after nrenaration. or, if necessary. after storage at -2O’C by the phenol method, basically as described by Kay & Cooper for lymphocytes 1141.‘Cold extracts were- carried- out at 0°C without -sodium lauryl sulphate (SLS) whereas the 40” and 60°C extracts were carried out in the presence of 0.5 % SLS. In each case the second phenol extraction was carried out at 0°C. After extraction, the RNA was treated with ribonuclease-free DNAase (Worthington) for 15 min at 0°C and reprecipitated with 2 vol of ethanol. RNA was separated into its components by polyacrylamide gel electrophoresis as described by Loening [15]. Differential centrifugation to fractionate the subcellular components was carried out under two different regimes in an effort to locate the cellular position of the RNA species in question. (1) This method was devised primarily for the preparation of ribosomes 1161.as follows: lvmnhocvtes were allowed to swell for-i0 min in medium LS; containing 10 mM NaCI, 20 mM Tris-HCI buffer (nH 7.5) and 1.4 mM MaCI,. They were then disrupted-by 15 strokes of the plugg& in a Dounce homogeniser with a clearance of 0.002 inch. The homogenate was centrifuged at 6 000 g for 6 min and the pellet retained, being referred to as ‘nuclear fraction’. The supematant was spun at 120 000 g for 1 h, giving the ribosomal pellet, together with a ‘supernatant’ fraction. (2) The second method used was designed to produce purified nuclei [17]. The lymphocytes were susnended in medium LS as described above and then broken by 100 to 200 strokes of the pestle in the tight-fitting Dounce homogeniser. The homogenate was centrifuged at 850 g for
L
+
Fig. I. Abscissa: mobility in gel; ordinate:
Az6,.
Scans of polyacrylamide gel electrophoresis of total cellular RNA. (A) Normal lymphocytes; (B) Lymphocytes from patients with chronic lymphocytic leukaemia. Low molecular weight peaks are labelled a to e. Peak a contains 5S ribosomal RNA. Gels were of 5 % acrylamide, run for 2 h at 5 mA/gel. 10 min, to give a crude nuclear pellet, which was purified by the method of Blobel & Potter [18]. The supematant was centrifuged at 12000 g for 20 min, to yield a ‘mitochondrial’ fraction, and subsequently at 120 000 g to yield ribosomal and supernatant fractions.
Results and Discussion Electrophoresis of RNA from CLL lymphocytes on 50 % polyacrylamide gels revealed a series of peaks of optical density in the 4-10s region of the gel. The peaks were present also in RNA from normal lymphocytes, but in much smaller amounts (fig. 1). These low molecular weight RNA species had similar electrophoretic mobilities to SnRNA reported in other cell types [2-111. However in CLL cells the peaks represented about 4% of the total RNA, considerably more than the 0.5 % or less reported elsewhere [2-II]. The low molecular weight peaks could be extracted from intact cells by O”, 40” or 60°C phenol extractions, with no apparent diffference in Exptl Cell Res 75 (1972)
538
Preliminary notes
efficiency. The peaks were found in all sixteen CLL patients studied, regardless of leucocyte count (10 000-250 000 cells/mm3) or stage of the disease. 2.4 % gels revealed no differences in ribosomal or transfer RNA between normal or CLL lymphocytes. To ensure that these large amounts of small molecular weight RNA did not represent an artefact of extraction, the following tests were carried out. (1) RNA was extracted both from freshly prepared and also from previously frozen lymphocytes, but in both cases the 4-10s region of the gel was identical; (2) lymphocytes were prepared from peripheral blood by the defibrination technique of Chalmers [12], as well as by the Ficoll/Triosil gradient method to ensure that the different techniques did not account for the differences between normal and leukaemic cells. In fact, the RNA appeared to be identical whichever cell separation technique was used. (3) Omission of the DNAase treatment during the extraction procedure did not affect the appearance of the low molecular weight RNA peaks [4]. The peaks were removed by RNAase treatment. (5) Omission of precautions against ribonuclease action during extraction, i.e. control of temperature, absence of bentonite from extraction medium, did not change the amount of material in low molecular weight peaks, suggesting that the material is not an artefact produced by breakdown of larger RNA molecules during extraction. These results indicate that these peaks are genuine RNA species and, in such quantities, are a characteristic of chronic lymphocytic leukaemia. To establish the cellular localisation of the peaks, the lymphocytes were fractionated by differential centrifugation. Since the RNA could be extracted at 0°C without SLS even from fresh tissue, it was thought that this indicated a cytoplasmic location [14] possibly as a ribosomal compoExptl Cell Res 75 (1972)
nent. Accordingly, a ribosomal pellet was prepared, and the RNA was extracted from this and from the other fractions produced during the preparation. Analysis of the RNA from each fraction on 5 % gels shows 5S RNA in the ribosomal fraction, but none of the other small molecular weight components. In fact, the fraction that spun down at 6 000 g contained the 4-10s components. This fraction probably consists mainly of nuclei. In an effort to confirm that these low molecular weight components are of nuclear origin, nuclei were prepared and purified as described. The nuclei obtained were divided into 2 batches, the first batch being extracted at 0°C without SLS, and the second at 60°C with 0.5% SLS. Both methods extracted the low molecular weight RNA components from the nuclei. Preparations of RNA from other cellular fractions, i.e. 12 000 g and 120 000 g pellets and 120 000 g supernatant show no signs of these components. Thus it appears that the low molecular weight RNA speciesfound in leukaemic lymphocytes are similar to the SnRNA previously reported from various tissues. Whether the presence of increased amounts of these nuclear components is related in any way to the inactive nature of the lymphocytes in CLL remains to be seen. It is hoped to investigate the kinetics of labelling of this fraction in both normal and CLL cells in an effort to further characterise it, and possibly to assign it a role in cell function. We wish to thank the Medical Staff of the Clinical Haematology Department, Manchester Royal Infirmary. and of the Radiotherapy and Pathology departm&ts of the Christie Hospital for their cooperation. The work was supported by a grant from the Leukaemia Research Fund.
References 1. Havemann, K & Rubin, A D, Proc sot exptl biol med 127 (1968) 668.
Preliminary notes 2. Hellung-Larsen, P, Tyrsted, G & Frederiksen, S, 3. 4. 5. 6. 7.
Abstract VII meeting of Nordic Society for Cell Research. Exptl cell res 67 (1971) 10. Howard, E & Stubblefield, E, Exptl cell res 70 (1972) 460. Weinberg, R, Biochim biophys acta 190 (1969) 10. Rein, A & Penman, S, Biochim biophys acta 190 (1969) 1. Busch, H & Smetana, K, The nucleolus (ed H Busch & K Smetana) pp. 285-317. Academic Press, New York and London (1970). Hodnett, J L & Busch, H, J biol them 243 (1968)
6334. 8. Prestayko, A W, Tonata, N & Busch, H, J mol
bio147 (1970) 505. 9. Prestayko, A W, Tonata, N, Carol-Lewis, B &
Busch, H, J biol them 246 (1971) 182. IO. Frederiksen, S, Tnnnesen, T & Hellung-Larsen,
P, Arch biochem biophys 142 (1971) 227. 11. Hellung-Larsen, P & Frederiksen, S, Anal biothem 40 (1971) 227. Harris, H & Ukaejiofo, E 0, Lancet 2 (1969) 237. ::: Coulson, A S & Chalmers, D G, Immunology 12 (1967) 417. 14. Cooper, H L & Kay, J E, Biochim biophys acta 147 (1971) 322. 15. Loening, U E, Biochem j 102 (1967) 251. 16. Kay, J E, Ahern, T & Atkins, N, Biochim biophys acta 147 (1971) 322. 17. Laszlo, J, Ta-Fu Huang, A & Kremer, W B, Methods in cancer research (ed H Busch) vol. 5, p. 377. Academic Press, New York and London (1970). 18. Blobell, G & Potter, V R, Science 154 (1966) 1662. Received August 8, 1972
Some characteristics of the morphogenic inhibitor in blastocoelic fluid from sea urchin embryo of Zoology, University of California, Berkeley, Calif. 94720 and Bodega Marine Laboratory, University of California, Bodega Bay, Calif 94923 USA W. E. BERG, Department
External application of the blastocoelic fluid from the sea urchin embryo was found to inhibit gastrulation reversibly [l]. The following are additional observations on the characteristics of the inhibitor. Blastocoelic fluid (hereafter referred to as BF) preparations were obtained, as previously described, by removal of the hyaline layer at the early gastrula stage and flattening the embryos by centrifugal force. The embryos
539
pack down and the clear viscous supernatant is presumed to be, in large part, fluid forced out from the blastocoel through the ruptured gastrular walls. An approximate assayfor the potencies of the preparations consisted of serial dilutions in microliter culture chambers. Test embryos for determining the degree of inhibition of morphogenesis were added at a late blastula stage. The original study was confined to embryos of Lytechinus pictus. Cross tests of BF solutions have now been carried out between the echinoderms L. pictus, Strongylocentrotus purpuratus, and Dendraster excentricus. No species specificity of BF from the 3 echinoderms was observed, although there was noticeable variability of the inhibitory potencies and the sensitivity of the embryos. In general, gastrulation in S. purpuratus embryos was more sensitive to the inhibitor and morphogenesis was blocked in relatively high dilutions of the various BF preparations. BF from Dendraster embryos was considerably weaker than from the other species; usually gastrulation of test embryos was very slowly completed leading to radially symmetrical postgastrulae. BF from L. pictus embryos had no specific morphogenic effect on gastrulation of Urechis caupo embryos whereas it had strong effects on development of embryos of Mytilus edulis and Ciona intestinalis. For example, M. edulis embryos at hatching exposed to a l/2 dilution of L. pictus BF generally disintegrated. In l/4 and l/S dilutions they remained motile and developed an apical tuft, after 48 h a velum formed but no shell and little sign of internal organization. Due to the opaqueness of the embryos, it was not possible to observe whether this inhibition of morphogenesis and differentiation stemmed from an initial effect on gastrulation. L. pictus BF also inhibited development of C. intestinalis embryos. In l/2 dilutions the embryos, added shortly before Exptl Cell Res 75 (1972)