Inhibition of chondrogenesis by normal mouse serum in cultured chick limb cells

Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved

0014.4827/R01110021-10$02.00/0

Experimental

INHIBITION

Cell Research 130 (1980) 21-30

OF CHONDROGENESIS

SERUM

IN CULTURED

CHICK

BY NORMAL LIMB

MOUSE

CELLS

CURTIS L. PARKER, DOUGLAS F. PAULSEN,’ JOSEPH A. ROSEBROCK and W. CRAIG HOOPER Department of Anatomy, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27103, USA

SUMMARY Chick limb-bud mesoderm cells from embryonic stages 22-25 were cultured at high cell densities in media known to support chondrogenesis. Under these conditions the continuous presence of normal mouse serum, at a concentration of lo%, inhibits the ability of the cells to produce toluidine blue-stainable cartilage matrix materials. In contrast, mesodermal cells treated with comparable concentrations of other heterologous sera continue to differentiate much like the control cultures while growing in the presence of the test sera. The inhibitory effect of the serum was shown not to be the result of a general cytotoxic effect on protein synthesis or the inability of the cells to incorporate [3H]D-ghtcosamine. There was a significant difference however, in the distribution of the incorporated glucosamine. Less label was associated with the cell layer of the treated series, while a greater amount of the incorporated material was found to be secreted into the medium when compared with the control cultures. Studies have shown also that the serum inhibitory response is dose dependent, while the factor(s) itself is non-dialysable, stable to heat and repeated freezing and is not a conventional serum lipoprotein. Following the addition of whole or delipidated mouse serum, a significant increase in lipid droplets appears in the cytoplasm of the cells. Biochemical analyses of mouse serum-treated cells indicate that there is a marked increase in their triglyceride content as compared to the control cells. While the nature of the serum inhibitory factor remains to be determined, the accumulation of triglyceride following mouse serum treatment suggests that this may play a role in modulating the expression of the chondrogenic phenotype.

It has been well documented that chick limb-bud mesoderm cells from embryonic stages 22-25 are capable of terminal differentiation in vitro when the dissociated cells are cultured at high cell densities [l-5]. Depending on the medium and cell density used, three major tissue types are produced in the cell cultures [Z, 51. These three tissues have been identified as skeletal muscle, cartilage and a connective tissue element represented primarily by a fibroblast-like cell type [2, 51. The two former tissue types can be readily identified with the inverted phase-contrast microscope. Skeletal muscle is recognizable following

the fusion of myoblasts to form multinucleated myotubes which undergo spontaneous contractions. The chondrogenic cells undergo a phase of aggregation (clustering of cells with a cell density greater than that of the surrounding areas), followed by the secretion of refractile extracellular matrix materials [3]. The cartilage matrix can also be stained with toluidine blue to detect metachromasia of the cross-linked matrix. The staining property of the glycosaminoglycans (GAG) ’ Present address: Department of Anatomy, Morehouse School of Medicine, Atlanta, GA 30314, USA.

22

Parker et-al.

secreted by the ~hondrobIasts was used to determine the effect of mouse serum on chondrogenesis while maintaining limb mesodermal cells in culture. The results reported in this communication indicate that normal mouse serum is capable of inhibiting d~~re~tiation of cultured limb cells. Further, mouse serum signi~~antly stimulates the accumulation of neutral lipids by the cells. These findings suggest that stimulation of triglyceride synthesis by the mouse serum may play a role in modulating the expression of the chondrogenic phenotype following the accumulation of lipid in the presumptive chondrogenic cell population.

MATERIALS

AND METHODS

Limb buds from stage 22-25 [6] White Leghorn chick embryos were used in these experiments. Sterile precautions were observed throughout the dissociation and culture procedures. The limb buds were dissected free and collected in a dish containing Tyrode’s solution. In must experiments, the limbs were cut into fragments prior to transferring to depression dishes containing 0.25% trypsin (Nutritional Biochemicalsf and 0.1% EDTA in Ca*+- and Mg+-free Tyrode’s solution. The limb-bud tissue was incubated in the trypsin-EDTA so&ion at 37°C for 45-60 min. Following incubation* the limb tissue was rinsed free of the trypsin-EDTA with complete medium. The tissue was dispersed in 2 ml of complete m~ium by agitation on a Vortex mixer to obtain a single cell suspension. The cell suspension was centrifuged through a sterile Nitex filter (pore size 20 pm) at 500 rpm to remove any remaining cell ciumps. The cell density of the filtered cell suspension was then determined using a hemocytometer. The cells were plated at a high density (approx. 5x lo6 cells) in 35 mm plastic tissue culture dishes (Falcon Plastics) containing 2 ml of complete medium. For some of the studies reported here, the cells were cultured by the micro-mass technique [3]. “The cultures were incubated at 3’i”C under a humidified atmosphere of 5 % CO, in air.

Crclfurr medirnm The culture medium consisted of Eagle’s MEM (Gibco) supplemented with 7 % horse serum (KC Biological), 3% fetal bovine serum (KC Biological), 5 % IO-day-old chick embryo extract, penicillin (100 Ui ml) and streptomycin (IO0 &ml). After allowing the cells to attach overnight. fresh complete medium containing an additional 10% of horse ierumlfetal bovine serum (7: 3) was added to the control cultures while 10% mouse serum or other test sera was added to the

exoerimentai cultures, The medium was then changed on’alternate days. At the end of the culture per&d+ the cells were rinsed in 0.9% saline, fixed in 95% ethanol-10% Bouin’s solution and then stained with 1% toluidine blue to show the cartilage matrix.

Tesf sera The test sera were obtained by exsanguination the animals in the laboratory. Sera from ICR-DUB mice purchased from Flow Laboratories, Dublin, VA, and B6D2F, mice from Jackson Laboratories, Bar Harbor, ME, w&e employed in these studies. The other animal sera were obtained locally and used prior to freezing. It has been reported [7] that rat embryos cultured in homologous serum which was prepared from blood allowed to clot overnight, prior to separation of the serum, led to m~formations in the developing rat heart. In contrast. no n~alformations were observed when the blood was centrifuged before clotting and the serum used, or when plasma was obtained from heparinized blood which was immediately centrifuged. In light of the previous report, mouse serum was analysed from blood centrifuged prior to and foilowing clotting. in addition, plasma was obtained by collecting blood in heparju-containing tubes which was immediately centrifuged. From these studies, it was found that there was essentially no difference in the effect on cultured cells regardless of the method of preparation of the test material. For this reason, most of the studies reported here were carried out using mouse serum which was pooled from a number of animals exsanguinated the same day and the blood allowed to clot overnight at S”C prior to centrifugation and sterile filtration.

The mouse sera were delipidated by the procedure of Rude1 et al. [S]. For this procedure approx. 8 ml of fresh sera were raised to a solvent density of 1.22 with solid KBr (0.351 g of KBrlml of serum). The serum (d 1 : 22) was then ptaced in an ult~centrifu~e tube and overlaid with additional d 1: 22 solution, which was prepared by the addition of solid JSBr to the buffered d 1.006 solution of Scanu & Granda [9]. The serum was spun at 200000 g for 24 h in a Beckman L2-65B ultracentrifuae using an SW40 rotor, This procedure renders betterthan 95% of the serum lipoproteins free at the top of the centrifuge tube, Following ultracentrifu~l flotation, the enriched lipoprotein layer (supernatant) and the remaining serum components (infranatant) were dialysed extensively at 4°C against several changes of 0.15 M NaCl, then sterilized by filtration. The volume of each was determined and adjusted so as to contain comparable concentrations of their constituents as those found in whole serum.

Tritiated leucine (spec. act. 54.6 CifmM) and [“I-I]D-~ncos~jne (spec. set. f0.13 Ci/mM) were purchased from New England Nuciear, Boston, Mass. The celis were pulse-la&fed at various periods of cul-

Inhibition of chondrogenesis by mouse serum

23

b Fig. 1. Toluidine blue-stained areas of cells cultured at high density by the micro-mass technique. (a) Control day 5; (b) 5-day mouse serum treated at 10%. In (a) note the large number of cartilage nodules represented by the dark-stained material which is sepa-

rated by internodular cells which do not stain. In (b) there are only faintly stained areas which are not metachromatic. The insets demonstrate entire micromass ‘spots’ X5.

ture with 10 /Xi [3H]1eucine. At the end of the labeling period the cells were washed 3x with ice-cold saline and scraped from the culture dishes with a rubber policeman. The cells were homogenized in a Potter Elvehjem tissue grinder (Fisher Scientific) fitted with a Teflon pestle. The homogenates were made 10% with respect to cold TCA and stored overnight at 5°C. The TCA-precipitable material was pelleted by centrifugation at 2500 rpm for IO min. The pellets were washed 3 times with 5% TCA and the final washed pellets were solubilized in 1 ml of 1 N NaOH. Aliquots of the solubilized cells were used for protein determination by the method of Lowry et al. [lo] using bovine serum albumin as a standard. Part of the remaining material was neutralized with I N HCI, then added to IO ml Scintiverse (Fisher Scientific) and counted in a Packard Tri-Carb liquid scintillation spectrometer. For the incorporation of [3H]D-glucosamine, the cells were pulsed with 3 &i of the isotope per ml of culture medium. Following the pulse, the medium was removed to determine the amount of secreted glycoprotein by TCA precipitation. The cells were washed with cold saline, homogenized, and TCA precipitated as described above. Aliquots were used for scintillation counting and protein determination.

through a graded series of ethanol and embedded in situ in Epon 812 [12]. Thin sections obtained with a diamond knife in a Sorvall MT 2B ultramicrotome were double-stained with lead citrate and uranyl acetate before viewing in a Philips EM 400 electron microscope.

Electron microscopy Cells for electron microscopy were prepared as previously described by Smith et al. [I I]. In brief, the cells were fixed in the culture dishes for 1 h with 3 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. The cells were post-fixed in the same buffer containing 1% osmium tetroxide overnight, dehydrated

Lipid analysis Cells (1 x lO’/culture) growing in 60 mm culture dishes were treated for 6 h with a final concentration of 10% mouse serum in Eagle’s MEM plus supplements. The cultures were then washed 3x with cold saline and the cells were scraped from the dishes with a rubber policeman and placed in 15 ml conical centrifuge tubes containing 1 ml methanol/water (2: I). The dishes were rinsed with an additional 1 ml of methanol/water which was added to the centrifuge tubes as well. The lipid was extracted by the procedure of Bligh & Dyer [13]. The extracted lipid was then used to measure the phospholipid phosphorus [ 141and triglyceride content [15] of the treated cells and compared with cell cultures handled in a similar manner but receiving an additional 10% mixture of horse/fetal bovine serum (7 : 3) in place of the mouse serum.

RESULTS Effects of mouse serum on limb cell chondrogenesis Our initial observation on the treated cells was that by 3 h following the addition of Exp Cell Res 130 (1980)

24

Parker et al.

Fig. 2. Chick limb cells cultured at 5x IV cells/35 mm dish in MEM plus supplements. The cells were allowed to attach overnight and the medium was changed. The following additions were then made: (a) MEM control containing an additional 10% horse serum/fetal bovine serum (7: 3); (b) MEM containing 10% guinea pig serum; (c) MEM containing 10% monkey serum; (d) MEM containing 10% mouse serum; (e) MEM containing 10% isolated mouse serum lipoproteins; cf) MEM containing 10% delipidated and dialyzed mouse serum. The cell cultures were fixed and stained after 5 days’ growth. Dark staining material represents toluidine blue-stained cartilage colonies. Note the lack of metachromatic cartilage matrix materials in (d) and (f‘).

normal mouse serum at a concentration of lo%, the cells began to accumulate dense granules which were readily apparent with the inverted phase-contrast microscope. These granules became somewhat more pronounced with time, otherwise the cells appeared normal. Further, on day 2 of culture with mouse serum there was a noticeable lack of cell aggregation (increased cell density in specific areas); a process which appears to be important for subsequent chondrogenesis to occur [3], and a lack of refractile matrix when compared with the amount seen in the untreated control cultures. With increased time in culture, the control cells accumulated more refractile cartilaginous matrix and many of the cartilage nodules underwent fusion, giving rise to larger nodular masses. This process remained conspicuously absent in the treated cultures even after 5 days of culE.rp Cd Res 130 (1980)

ture. The lack of significant amounts of cartilage matrix was most evident following fixation and toluidine blue-staining of the cells. Cells cultured by the micro-mass technique [3] showed faintly stained areas of the treated-cell culture surface. These areas did not appear metachromatic. In contrast, the control cultures contained large discrete metachromatic nodules of cartilage throughout the micro-mass ‘spot’ (fig. 1). Ejfect of other heterologous sera and delipidated mouse serum on limb cell chondrogenesis

In order to determine whether the inhibitory effect on chondrogenesis was an exclusive property of mouse serum, other heterologous sera were also tested. Several sera including human, monkey, rat and guinea pig were analysed. In addition, mouse serum was delipidated and both the lipoprotein and delipidated fractions were tested on cultured limb cells. Some of the results are presented in fig. 2. None of the other sera listed above demonstrated a marked effect on chondrogenesis, nor did any of them produce granule formation which could be detected with the inverted phase-contrast microscope. Similar results were obtained using the lipoprotein fraction of the mouse serum (fig. 2e). It was found, however, that the serum infranatant (delipidated fraction) was as inhibitory as whole mouse serum (fig. 2j; d, respectively; compare also with fig. 2a, b, c, e). This result suggests that the serum inhibitory factor is not a conventional serum lipoprotein. Dose-dependence of mouse serum

and other properties

In order to establish whether or not the inhibitory effect of mouse serum was con-

Inhibition of chondrogenesis by mouse serum

25

Fig. 3. Effect of various doses and heat inactivation of mouse serum on limb cell chondrongenesis. The cells were plated at 5x lo6 cells/dish and allowed to attach overnight. The cells were then grown continuously in MEM plus supplements for 6 davs with the following additions: (a) Control containing 10% horse serum/fetal bovine serum (7 : 3): (b) 1% mouse serum; (c) 2% mouse serum; (d) 4% mouse serum; (e) 6% mouse serum; cf) 8 % mouse serum; (g) 10% mouse serum; (h) 10% heat-inactivated (56°C for 60 min) mouse serum. Dark-staining material represents metachromatic cartilage colonies.

centration dependent, a study was undertaken using the same lot of serum at various concentrations. The results are presented in fig. 3. These results clearly show that there is a concentration effect on

Table 1. Incorporation of [3H]leucine bJ cultured chick limb mesoderm cellsa

Day 1

Pulse length (hours)

Control (vdmg protein)

Mouse serum treated (cpmlmg protein)

1 2 3 1

7 184 17 021 21 065 6 624 12 242 20 251 6 427 14 232 27 076

6 567 13 834 19 849 6 065 12 999 20 91.5 4 313 10 750 13 207’

2

3*

3? 1 2 3

a The limb cell cultures were set up with approx. 5~ 10” cells/35 mm culture dish and allowed to attach overnight. The next day (day 1) the treated cultures received fresh complete medium containing 10% mouse serum. The control cultures received fresh complete medium containing an additional 10% horse/ fetal bovine serum (7 : 3). The cells were pulse-labeled with 10 /.&I of [3H]leucine for 1, 2 and 3 h each day for 3 days and processed as described in Materials and Methods. Each count is the average of duplicate samples. The data represent one of three replicate experiments. b Chondrogenesis was occurring in the control cultures at this time. c Significantly different from the control at p
chondrogenesis when the cells are grown continuously in the presence of various amounts of mouse serum. There were slight variations in the amount of cartilage produced with different lots of sera from the same or different mouse strains. Sera from five different strains have been tested (unpublished observation) and all were totally effective in inhibiting chondrogenesis when added to the culture medium at a concentration of 10% (fig. 3g). To rule out the possibility that the inhibitory effect was due to a complementmediated immunological response, mouse sera were heated at 56°C for 60 min. These sera were found to be just as inhibitory following heat inactivation (fig. 3 h). Similar results were obtained with repeated (5x) freezing of the sera in liquid nitrogen followed each time by thawing in a 37°C water bath (data not shown). Likewise, extensive dialysis of the sera from both ICRDUB and B6D2F, mice did not alter the inhibitory effect on chondrogenesis as seen in fig. 2f for dialysed delipidated serum. Effect of mouse serum on macromolecular synthesis by chick limb cells An attempt was made to determine if the inhibition of chondrogenesis might be due to a decrease in protein synthesis or hexose utilization by the treated cells. The results Exp CdRes

130 (1980)

26

Parker et al.

Table 2. Incorporation of [3H]~-glucosamine by cultured chick limb mesoderm cellsa Glucosamine Condition of cultures Control Mouse serum treated

Cell layer Wdmg protein)

Total cpm

Medium (cpmlmg protein)

11 948f491

17 247

5 299k845

17 107

8 384k679'

8 723+ 86b

a The limb cell cultures were set un at approx. 5x 10” cells/35 mm culture dish. The mouse se&m (10 %) and additional 10% horse/fetal bovine serum (7: 3) were added after the cells were allowed to attach overnight. The cells were maintained for 4 days, at which time the control cultures were undergoing extensive chondrogenesis. The cultures were pulselabeled on day 4 with 6 pCi [3H]D-ghrcosamine for 5 h, and the medium and cell layer analysed as described in the Materials and Methods. The data represent the mean k S.E.M. for 3 experiments. b Significantly different from the control at ~~0.05 level, Student’s r-test. p Significantly different from the control at p
indicate that leucine incorporation is linear during the 3-h labeling period and that there is essentially no difference in the amount of TCA-precipitable [3H]leucine incorporated by the treated cells when compared with their paired controls during the first 2 days of culture. It is interesting to note, however, that there was a significant decrease in the amount of [3H]leucine incorporated by the treated cells after this time (table 1). It should be noted also that this is the period during which the control cells are undergoing chondrogenesis; a process which appears to be suppressed in the treated cultures. Table 2 shows the result of [3H]D-glucosamine incorporation into cellular glycoproteins and secreted materials. It can be seen that mouse serum increases the amount of labeled hexose-containing protein released into the culture medium durEXP Cell Res 130 (1980)

ing cartilage development. The increase in soluble products may indicate a lack of organization of the matrix in the treated cells and thus a lack of chondrogenesis. This is suggested by the results, since there is essentially no difference in the total amount of label when the counts from the cell layer are combined with the secreted soluble counts (table 2). The data from tables 1 and 2 further indicate that mouse serum does not have a general cytotoxic effect on the cells. Changes in the lipid content of limb cells following serum treatment For this series of studies cells were plated at a low cell density (-5x 105/35mm dish). This cell density normally does not give rise to cartilage colonies even after extensive periods in culture [ 1,4]. However, since the same morphological changes occur at both low and high cell densities, a low cell density was used so that individual cells could be observed. The cultures were maintained overnight and one series of cells then received mouse serum at a concentration of 10%. The other series of cells received an additional 10% horse serum/fetal bovine serum (7 : 3) and both series were maintained in culture for an additional 24 h. The cells were then fixed and stained with Oil Red 0 to determine if the dense droplets were indeed lipid. In addition, cells treated for a comparable period (24 h) were rinsed in physiological saline, fixed with glutaraldehyde, post-fixed with osmium tetroxide and prepared for electron microscopy [ 111. Fig. 4 illustrates the large amounts of what is believed to be lipid which accumulates in response to the added mouse serum. Small amounts of lipid are also seen at either microscopic level in the control cells. Therefore, the mouse serum appears to

inhibition of chondrogenesis by mouse serum

Fig. 4. Chick’ limb mesoderm cells grown in MEM plus supplements. The following additions were made during the last 24 h of culture: (a) 10% horse/fetal bovine serum, x5000; (b) 10% mouse serum, x4000. Note the large amount of accumulated lipid in (b)

27

(arrowheads). The inset in (b) is a light micrograph of chick limb cells stained with Oil Red 0. The darkstaining granules are Oil Red O-positive lipid droplets. x200.

Exp Cell RPS I30 f 1980)

28

Parker et al.

Table 3. Phospho~~p~d phosp~z~ru~ and triglyceride context of cultured chick Ii~~ cells” Condition of culture

nmol phospholipid phosphorus/ mg protein

nmol triglyceride/ mg protein

to an increased deposition of triglycerides (triacylglycerols), the major form of stored lipids in animal cells. DISCUSSION

Several investigators have reported on the conditions (cell density, stages and media) necessary to obtain chondrogenesis by cul154.82 14.2 151St 8.9” tured limb mesoderm cells [l-5]. In this a The limb cell cultures were set up at approx. I x LO’ report we have shown that normal mouse celtsl60 mm culture dish. The mouse serum was added serum has a significant inhibitory effect on to the treated cultures at a concentration of 10% and the control cultures received an additional 10% the chondrogenic potential of cells which horse/fetal bovine serum (7 : 3) for 6 h. Following the treatment period the cells were washed with saline and otherwise are capable of high levels of the lipid extracted by the method of Bliah & Dyer chondrogenesis. In addition, the inhibitory [lo]. The phospholipid phosphorus was m>asured.by response appears to be a property of mouse the method of Chalvardiian rll] and the triglyceride content was measured by the-procedure of Sardesai & serum exclusively, at least among the variMatmine T121. Each value renresents the mean+ ous sera that we tested, since none of the S.E.M.?oi thiee experiments done in duplicate. ” Significantly different from the control at p
145.0f 5.1

E.rp Cell Res 130 (1980)

53.9+ 12.0

Inhibition

indicate alterations in phospholipid synthesis by the treated cells following a short pulse with a mixture of radioactive fatty acids; a full report on the total lipid changes in the cells following mouse serum treatment will be published elsewhere [22]. Labeling experiments utilizing [3H]leucine suggest that the inhibitory effect of mouse serum is not a general cytotoxic effect, since the isotope is incorporated as well by the treated cells as the controls during the period prior to overt chondrogenesis. The decrease in protein synthesis in the treated cells following this time is believed to be related to the lack of synthesis of stainable extracellular cartilage matrix. The inhibitory effects of vitamin A on chondrogenesis in mouse and chick embryonic tissues have been well documented [23-261. Some of the observed changes are similar to those reported here. Solursh & Meier [23] reported an accumulation of lipid droplets in chick chondrocytes following a 24-h treatment with vitamin A; much like our finding which occurs much earlier. Hassell et al. [24] reported that vitamin A had little effect on cell growth when the cells were cultured at low density. We found a similar situation with less than confluent cell densities (data not shown). Further, De Luca et al. [25] presented evidence that vitamin A may serve as a lipid intermediate in glycoprotein synthesis. While this work was in progress, a report appeared in the literature dealing with the effect of mouse serum on other cell types [27]. The authors did not present definitive data on the nature of the lipid changes in the cells: however, they did suggest that mouse serum induced adipose conversion of the various cell types studied [27]. This recent article corroborates much of what we report here as well as our findings on lipid accumulation in Rhesus mon-

of chondrogenesis

by mouse serum

29

key smooth muscle cells and skin fibroblasts (unpublished observations). The nature of the inhibitor factor is under investigation in our laboratory. The mechanism by which the mouse serum modulates the expression of the chondrogenie phenotype is unknown. This mechanism, however, may involve an inability of the secreted materials to become organized in the extracellular matrix thereby leading to the increase in TCA-precipitable soluble materials in the culture medium of mouse serum-treated cells (table 2). Finally, while the accumulations of lipid may or may not play a direct role in the inhibition of chondrogenesis, it is reasonable to speculate that altered lipid metabolism may render the chick cells incapable of functioning as chondroblasts. We wish to thank Dr L. Rude1 for his assistance in delipidating the mouse sera and Dr M. Waite and MS P. Sisson for the lipid analyses. This work was supported in part by a grant to C. L. P. from the General Research Support Grant RR-5404 from the NIH.

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Parker et al.

15. Sardesai, V M & Manning, J A, Clin them 14 (1968) 156. 16. Granstrom, M, Exp cell res 82 (1972) 426. 17. Beran, M, Exp hematol2 (1974) 58. 18. Metcalf, D & Russell, S, Exp hematol4 (1976) 339. 19. Curtiss, L & Edgington, T S, J immunol 116(1976) 1452. 20. - Ibid 118 (1977) 1966. 21. Chapman. H A, Jr & Hibbs, J B, Jr, Science 197 (1977) 282. 22. Parker, C L, Waite, M & King, L, Biochim biophys acta (1980). In press. 23. Solursh, M & Meier, S, Cal tiss res 13 (1973) 131.

24. Hassell, J R, Pennypacker, J P & Lewis, C A , Exp cell res 112 (1978) 409. 25. De Luca, L M, Silverman-Jones, C S &Barr, RM, Biochim biophys acta 409 (1975) 342. 26. Pennypacker, J P, Lewis, C A & Hasseil, J R, Arch biochem biophys 186 (1978) 351. 27. Maeda, Y Y, Rokutanda, M & Yamoto, N 3 EXP cell res 126(1980) 99. Received November 21, 1979 Revised version received June 23, 1980 Accepted June 24, 1980

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