Phytohemagglutinin stimulation of human lymphocytes

(:LIMCAL

IIIXIUNOLOGY

AND

Phytohemagglutinin Failure

3,353-362

IMMUNOPATHOLOGY

Stimulation to Detect Cyclic

AMP

(1975)

of Human

an Early

Increase

lymphocytes in

Concentration

ARTHUR GLASGOW, PETER POLGAR, INNA SAPOROSCHETZ, HWAKYU KIM, ALEXANDER M. RUTENBURG, AND JOHN A. MANNICK Depurtment

of Surgery, Boston.

Boston Unicersity Medical Massachusetts 02118

Center,

AND SIDNEY Department

of Medicine, Boston,

R. COOPERBAND Boston University Medical Massachusetts 02118

Received

March

Center,

27, 1974

Cyclic AMP content and adenylate cyclase activity were determined in human lymphocytes stimulated to proliferate by phytohemagglutinin (PHA). Both commercial (Difco PHA-M) and purified PHA were used at optimal stimulatory concentrations as confirmed by incorporation of [‘Tlleucine. Cyclic AMP levels were measured by radioimmunoassay. Exposure of lymphocytes to PHA-M alone yielded no significant change in intracellular cAMP levels; however, in the presence of the phosphodiesterase inhibitor, caffeine, PHA-treated cells had lower levels of CAMP than the caffeine controls. That adenylate cyclase remained active in these experiments was shown by marked increases in lymphocyte CAMP levels following exposure to prostaglandin E, (PGE,). Stimulatory concentrations of PHA-M or purified PHA also failed to induce adenylate cyclase activity in lymphocyte particulate preparations whereas PGE, or NaF produced significant increases in enzyme activity. Moreover, PGE,, at concentrations which caused a prompt and significant rise in adenylate cyclase activity and in intracellular CAMP levels, inhibited lymphocyte stimulation by PHA.

INTRODUCTION The role of cyclic adenosine 3’,5’-monophosphate (cAMP) in lymphocyte stimulation is controversial. On the one hand, Smith et al. (1,2) reported that exposure of human lymphocytes to phytohemagglutinin (PHA) produced marked early increases in intracellular CAMP levels which appeared to precede other well-known metabolic changes which accompany PHA stimulation. This report of an association between lymphocyte stimulation and an early rise in CAMP levels was supported by Ishizuka et al. (3) who found that CAMP and dibutyryl CAMP increased antibody formation by mouse spleen cells in response to sheep red blood cells both in vitro and in uivo. This finding was further supported by Hirschhorn et al. (4) and by Whitfield et al. (5) who reported that lymphoid cells maintained in culture could be stimu353 Copyright All rights

@ 1975 by Academic Press, Inc. of reproduction in any form reserved.

354

GLASGOW

ET

Al..

lated to proliferate by low concentrations of cAMP in the culture medium, However, Smith et ~2. (2) also found that compounds that increased intracellular CAMP levels inhibited lymphocyte stimulation by PHA, as did the addition of dibutyryl CAMP to the culture medium. Hirschhorn also reported that CAMP, theophylline, and dibutyvl CAMP inhibited PHA stimulation of human lymphocytes (4). The possibility that lymphocyte activation may be inhibited by increased intracellular levels of CAMP is also suggested by recent work of Henney and co-workers who reported that adenylate cyclase stimulators, phosphodiesterase inhibitors, cAMP and dibutyryl CAMP all inhibited the cytolytic activity of an immune population of mouse lymphocytes (6). Moreover, Novogeodsky and Katchalski (7) reported that doses of PHA which produced cell transformation in rat lymph node lymphocytes did not produce a rise in CAMP levels or in adenylate cyclase activity. Makman (8) has also reported that doses of PHA known to produce transformation when added to intact mouse thymocytes decreased the amount an adenylate cyclase activity in the cell lysates. When PHA was added to cell lysates, it had no effect on adenylate cyclase activity. Hadden (9) has also confirmed that mitogenic concentrations of PHA had no effect on cAMP concentrations in human peripheral lymphocytes. Because of uncertainty as to the relationship between lymphocyte stimulation and intracellular CAMP levels, we have re-examined the cAMP levels and adenylate cyclase activity of human lymphocytes stimulated to undergo proliferation by PHA. Portions of this work have been previously reported in abstract form (10). MATERIALS

AND

METHODS

Isolution of lymphocytes. Venous blood (100 ml) from normal human volunteers was drawn into heparinized syringes. The red cells were allowed to sediment by gravity for 2 hr at room temperature. The plasma was then aspirated and applied to columns of sterile cotton wool. After 15 min the lymphocytes were eluted from the columns with I50 ml of Eagle’s minimal essential medium (MEM). The cells were collected by centrifugation at 2OOg for 10 min, washed twice with MEM, recentrifuged at 200g for 10 milr then counted in a hemocylometer. The nucleated cells were 96-99% lymphocytes and were greater than 95% viable as judged by trypan blue dye exclusion. There were essentially no platelets and less than 1% of cells were red blood cells. Culture medium and reagents. The culture medium for all lymphocyte stimulation experiments was MEM containing 0.04 A4 glutamine, Id% fetal calf serum, penicillin 100 units, and streptomycin 100 kg/ml. Phytohemagglutinin-M (PHA-M) was obtained from Difco Labs., Detroit, MI and diluted in 5 ml of media prior to use. Purified PHA was prepared from Phaseolus Vulgaris (red kidney beans) by a method described elsewhere (11). In brief, the PHA preparation obtained from the precipitation of the mitogen from an acidified (pH 5.6) aqueous solution was purified further

PHYTOHEMAGGLUTININ

AND

LYMPHOCYTE

CYCLIC

AMP

355

by preparative polyacrylamide electrophoresis. This latter procedure yielded two homogenous bands. Their mitogenic activity was established by evidence of lymphocyte activation. This fraction was then used for the experiments with purified PHA described below. Prostaglandin E, (a gift of Upjohn and Company) was dissolved in distilled water containing a trace of Na,CO, and the pH adjusted to 7.0. The final concentration for PGE, was 5 X lo-” M. [ICJIeucine (1 mCilmM) was obtained from the New England NucIear Corporation. Lymphocyte stimulation-Leucine Incorporation Studies. All cultures contained 10 x 10” column purified lymphocytes/ml of culture medium in 13 mm x 100 tubes. [14C]leucine was added to a concentration of 0.25 &i/ml. Varying amounts of PHA-M were added and the cultures were incubated in a 95% air, 5% COZ, water-saturated atmosphere. After 6 days, 0.1 ml of bovine serum albumin (50 mglml) and 4 ml of 0.3 M perchloric acid (PCA) containing 10% casein amino acid (“casamino acids,” Difco Labs., Detroit, MI) were added to each culture. After thorough mixing, the tubes were centrifuged at 9OOg for 10 min. The supernatant was removed and an additional 4 ml PCA-casein amino acid solution was added. The centrifugation was repeated and the supematant was again removed. Four milliliters of 0.3 M PCA was again added. The mixture was heated to 60°C in a water bath for 15 min, and recentrifuged at 9OOg for 10 min. The supematant was decanted. One milliliter of 0.1 N sodium hydroxide was added to each tube. Finally, 5 ml of scintillation fluid prepared from dioxane naphthalene, PPO was added, and the tubes were counted in a Packard Tricarb liquid scintillation counter. Cyclic AMP determination. A quantity of 10 X 10” column-purified lymphocytes was added to test tubes containing 1 ml of culture medium preheated to 37°C in a water bath and PHA-M, PGE1, or caffeine. After addition of cells, the tubes were incubated for varying periods of time at 37°C in a CO, incubator as previously described. The incubation was terminated by the addition of 70% PCA (0°C) which contained 0.1 pmoles L3H]cAMP, to a final PCA concentration of 3.5%. The tubes were then centrifuged at 3OOOg, the pH of the supernatant adjusted to 7.0 with 3.0 N KOH and the precipitate removed by centrifngation. The supernatant was passed through a Dowex 50 W-X2 (hydrogen form) column and eluted with distilled H,O. The eluate was concentrated by lyophilization and the recovery (85%) of cAMP monitored with the added L”H]cAMP by counting an aliquot of the sample in a Packard Tricarb liquid scintillation counter. The amount of CAMP in the lyophilized material was determined by dissolving it in 300 ~1 of 0.01 M cacodylate buffer, pH 6.2 containing caffeine (2 PM) added to inhibit possible traces of phosphodiesterase in the assay mixture. ‘*“I-labeled cyclic nucleotide derivative (100 ,ul) and rabbit anti-CAMP antibody (100 ~1) each in cacodylate buffer, were added and the reaction mixture was incubated for 3 hr at 4°C. The antiserum was raised in rabbits using as antigen human serum albumin-O”‘-succinyl-CAMP conjugate prepared as described elsewhere (12,13). The antiserum produced was more

356

GLASGOW

“I

ET

10 CAMP,

AL.

10

J 100

pmoler

FIG. 1. Standard CAMP concentration curve. Each tube contains a standard quantity of cyclic AMP in 300 ~1 cacodylate buffer (0.01 M, pH 6.2), 2 PM caffeine, 100 ~1 ‘2”I-labeled cyclic nucleotide derivative diluted to contain 1.2-1, 8 x 10 CPM, and 100 ~1 rabbit anti-cAMP antibody; an excess of goat anti-rabbit globulin was added and the assay completed as described in the text. Each point represents the average and the vertical lines represent the range of three different determinations.

specific for cAMP than other nucleotides by a factor of 2 x 10’. ‘““I-labeled CAMP derivative was prepared by iodination of OZ’-succinyl-CAMP tyrosine methyl ester by the chloramine-T procedure of Hunter and Greenwood (14) and purified by chromatography on a Sephadex G-25 column as described elsewhere (12,13). After the 3-hr incubation an excess of goat anti-rabbit gammaglobulin (Miles Laboratories) was added. After 16 hr at 4°C total counts in each tube were determined (Nuclear Chicago automated gammawell counter). After addition of 3 ml of 0.05 M cacodylate buffer, the precipitates were isolated by centrifugation at 3400g at 4°C for 30 min and washed with cacodylate buffer. Radioactivity in the precipitates was determined and CAMP concentrations calculated with the aid of a standard curve (Fig. 1). The assay as described was sensitive within the range of 0.2-100 pmoles of cAMP with a maximum total error of &100/o. With this method it was possible to differentiate CAMP from other nucleotides by means of phosphodiesterase treated controls (13). Adenylute cyclase determination. Column purified lymphocytes (10 x 10”) were disrupted by exposure for 15 min to a hypotonic solution of 0.25 M sucrose containing 0.003 M glycylglycine buffer pH 7.4 and 1 mM MgSO,, followed by standardized homogenization in a tight pestle Dounce homogenizer at 4°C (13). The homogenate was then centrifuged at 3000g. The pellet containing nuclei and membrane material was washed in glycylglytine buffer, recentrifuged, and resuspended in the same buffer solution. Aliquots of this particulate preparation equivalent to 10’ whole lymphocytes and containing approximately 100 pg protein as determined by Lowry (15) method were incubated at 37°C for 15 min in 0.08 M Tris HCI buffer (pH 7.4) which contained 0.007 M MgSOJ, 0.004 M ATP, and 0.013 M caffeine. The various additions were (purified) PHA lO/pg, NaF (lo-.‘), PGE, (5 X lo-” M) or PHA-M 820 Kg, respectively. After 15 min cAMP levels were measured by radioimmunoassay as described above.

PHYTOHEMAGGLUTININ

AND

LYMPHOCYTE

CYCLIC

AMP

357

RESULTS Stimulatoy concentration of PHA. Since all prior lymphocyte stimulation experiments in our laboratory had been performed with 1 X 10” lymphocytes/ml, an initial experiment was undertaken to determine the concentration of PHA-M which would produce maximal stimulation of cultures containing 10 x lo6 lymphocytes/ml. The results of these experiments are shown in Table 1. It was apparent that 0.05 ml (820 pg) of PHA-M produced maximal incorporation of [‘4C]leucine into PCA precipitable protein. Similar maximal incorporation could be achieved with 10 pg of purified PHA. Consequently in all subsequent experiments designed to study the effects of PHA stimulation on CAMP levels or adenylate cyclase activity these concentrations of PHA-M or PHA were utilized. EfSect of PHA stimulation on lymphocyte CAMP levels. Because others (1,9) had reported an early increase in intracellular CAMP in human lymphocytes exposed to PHA with the maximum increase developing between 5 and 10 min after exposure of the cells to the mitogen, we studied the effects of incubation of lymphocytes with PHA-M, PGE,, caffeine, or a combination of caffeine and PHA-M in multiple experiments 2 and 7 min after initiation of the cultures. The results of these studies are shown in Table 2. It is apparent that exposure of lymphocytes to a concentration of PHA-M previously shown to be maximally stimulatory caused no significant change in intracellular CAMP levels after the 2 or the 7 min interval. On the other hand, in these same experiments, at the same time intervals there was a significant increase in intracellular CAMP levels in lymphocytes exposed to PGE1, an adenylate cyclase stimulator, or to caffeine, a phosphodiesterase inhibitor. To rule out the possibility that PHA-M directly stimulated adenylate cyclase and that the resultant increase in CAMP levels might have been negated by an increase in phosphodiesterase activity, caffeine was added to one set of tubes containing PHA-M in each experiment. It is apparent that the intracellular CAMP levels in these cells were no greater than those induced by caffeine alone, and in fact were less.

[%]LEUCINE

INCORPORATION

TABLE 1 BY 10 x 10" LYMPHOCYTES Culture

Control 0.025 ml PHA-M 0.05 ml PHA-M 0.075 ml PHA-M

cpm

(410 pg) (820 pg) (1230 Fg)

6157 14681 19573 15615

AFTER

6 DAYS'

CULTURE"

‘% -+ t r k

1391 2367 3005 1104

” Various quantities of PHA-M were added to cultures containing 10 X 10” column purified human lymphocytes and [“Clleucine (0.25 &i). After 6 days’ ‘“C incorporation into PCA precipitable protein was determined (see text). Results are reported as mean of twelve determinations k SD from three separate experiments performed in quadruplicate.

358

GLASGOW

EFFECT

Expt 2 min

7 min

incubation 1 2 3 4 5 6 7 8 9 10 11

Mean

~?r SE

AMP

I,E:vE:I,~

Caffeine (lo-‘M)

14.2 14.2 5.9 8.2 10.3 10.3 10.6

18.6 13.8 21.2 14.9 34.0 28.6 34.5

12.3 17.4 18.9 19.0 26.0 C2.6 33.85

17.0 11.1 18.9 18.1 10.3 10.3 10.6

10.0 k 1.25

10.5 * 1.3 P 10.7

23.7 -t 4.3 P < 0.01

22.7 _t 3.5 P i 0.01

13.2 2 1.7 P C’ 0.05 P cc 0.05”

11.6 16.8 15.3 10.9 11.3 16.9 17.3 20.2 18.2 19.5 16.4

12.8 16.8 15.4 9.6 15.5 24.5 19.5 10.5 21.9 19.5 10.3

15.8 k 1.05

16.0 k 1.6 P > 0.9

-.

PHA-M (0.05 ml)

<:YC~.IC

YGE, (5 x 10-s M)

incubation 1 12.6 14.0 2 3 5.9 4 8.0 5 9.3 10.2 6 7 9.2 k SE

Al,.

TABLE 2 AGENTS ON LYMPHOCYTE (PMOI,ES/~~’ CELLS)”

OF VAHIOUS

Control

Mean

ET

” Each value is the mean value * Compared to caffeine control.

207.0 133.1 196.3 66.1 26.5 26.6 29.9 29.8 55.9 32.5 43.7

of duplicate

76.9 r 29.3 P < 0.05

31.7 18.7 13.9 47.2 103.2 22.6 17.8 19.9 48.4 48.5 53.1 39.6 2 7.8 P < 0.01

Caffeine + PHA-M

(10 ? M) (0.05 ml)

23.0 22.6 22.0 36.9 50.6 12.8 14.2 13:f 33.8 35.6 45.8 28.2 _t 3.4 P cc 0.05 P c’: 0. I h

determinations.

As a further control to establish that PHA-M was indeed stimulating the cells, tubes in each of two of the experiments reported in Table 2 were incubated with [r4C]leucine for 6 days, and the degree of 14C incorporation measured at the end of that time period. In the first of these experiments, the PHA-M stimulated tubes had a mean of 40,450 cpm as compared with 1360 cpm in unstimulated controls. In the second experiment, the PHA-M stimulated tubes had a mean of 18,390 cpm as compared with 4890 cpm in control tubes. We, therefore, felt confident that the CAMP determinations were performed under conditions in which PHA-M stimulation of lymphocytes had taken place. In additional experiments CAMP levels were measured after longer periods in culture. A series of these experiments is illustrated in Fig. 2. It is again apparent that early after the establishment of the culture there was no difference between CAMP levels in PHA-M stimulated and control cells. At

PHYTOHEMAGGLUTININ

n.

AND

LYMPHOCYTE

TIME

(MINUTES)

CYCLIC

359

AMP

A

2

FIG. 2. A plot of the mean of multiple times after establishment of lymphocyte kg) PHA-M. The results are expressed ments performed.

determinations cultures with +-SE. Numbers

of lymphocyte CAMP levels at various and without the addition of 0.05 ml (820 in brackets are the number of experi-

time intervals the CAMP levels in the PHA-M stimulated lymphocytes appeared to be slightly lower than those of the unstimulated cultures; however, this had not yet reached the level of statistical significance 90 min after initiating the cultures. Efict of PGE, on lymphocyte stimulation by PHA. Because the preceding experiments had demonstrated that PGE, as opposed to PHA, produced a marked early increase in intracellular CAMP levels in human lymphocytes, we studied the effect of various concentrations of PGE, on [‘4C]leucine incorporation in cultures containing 10 X lo6 lymphocytes and the standard amount (0.05 ml) (820 pg) of PHA-M. The results are listed in Table 3. It is apparent that PGE, at all concentrations inhibited PHA stimulation. Effect of PHA on lymphocyte adenyl cyclase activity. In order to deterlater

TABLE

3

EFFECT OF PGE, ON LYMPHOCVTE STIMULATION Cpm

[ ‘“Clleucine Control

Cells PGE, PGE, PGE, PGE,

alone (5 x (1 x (1 x (I x

lo-” lo-” 1O-L lo-’

M) M) M) M)

814 326 638 1001 696

after

6 days’ PHA-M 8350 570 3218 4584 4001

BY PHA"

culture Percentage

suppression 0 100 68 50 58

” PGE, at various concentrations was added to cultures containing 10 X log column purified human lymphocytes, 820 pg PHA-M and [Wlleucine (0.29 pg). After 6 days’ ‘C incorporation into PCA-precipitable protein was determined (see text). Results are expressed as mean cpm of triplicate determinations.

360

Experiment

GLASGOW

with

commercial

PHA-M

Adenylate

cyclase

ET

activity/CAMP

AL

(pmoles/lOO

pg protein)

Experiment Addition None NaF (10 ‘! M) PHA-M (crude) PGE, (5 x lo-” Experiments

1 280 625 285 1135

820 pg M) with

purified

2

3

4

Mea11

171 202 224 209

115 335 202 487

106 148 67 -

168 328 195 610

~+ 77 ? 199 t 86 ir 426

P - 0.05 P i 0.25 P -: 0.01

PHA

Adenylate

cyclase

activity/cAMP

(pmoles/lOO

p.g protein)

Experiment Addition None NaF (lo-’ M) PHA-purified 10 pg PGE, (5 x 1O-5 M)

1

2

3

4

5

6

551 1113 468 1362

451 1695 220 1768

564 1077 936 489

435 580 400 590

612 1382 603 1313

286 437 296 468

Mca~r 483.2 1047.3 487.2 998.3

t 118 _‘475 f 257 +- 553

P <: 0.05 I’ 1 0.25 P -< 0.05

a Effect of commercially available and purified PHA on lymphocyte adenylate cyclase activity. Cyclic AMP levels were measured by radioimmunoassay in particulate lymphocyte adenylate cyclase preparations following incubation (15 min) at 37”, with PHA, NaF, and PGE,, respectively.

mine whether or not PHA had an effect on adenylate cyclase activity in a particulate crude lymphocyte membrane fraction, both PHA-M and the electrophoretically purified PHA were incubated for 10 min at 37°C with 100 pug of this particulate fraction. As positive controls sodium fluoride (lo-’ M) and PGE, (5 x lo-” M) were similarly incubated with the adenylate cyclase preparation. The results are shown on Table 4. It is apparent that neither the crude nor the purified PHA had any significant effect on lymphocyte membrane adenylate cyclase activity, whereas sodium fluoride and PGE, produced moderate to marked increases in this activity in every experiment but one. DISCUSSlON To determine if human lymphocyte proliferation is mediated by an intracellular increase in cyclic AMP or an increase in adenylate cyclase activity these cells were stimulated by doses of PHA-M or purified PHA shown to produce proliferation under the same culture conditions as those used for the nucleotide assay.

PHYTOHEMAGGLUTININ

AND

LYMPHOCYTE

CYCLIC

AMP

361

These studies failed to demonstrate any early or late rise in either CAMP levels or in adenylate cyclase activity in human lymphocytes stimulated to undergo proliferation by PHA. These results are at variance with those reported by Smith et al. (1) who found that exposure of human lymphocytes to high concentrations of PHA produced a significant early increase in intracellular CAMP concentration and stimulated lymphocyte adenylate cyclase activity. One possible explanation is that Smith et al. (2) exposed their lymphocyte preparations to a concentration of approximately 400 pg of commercial (Difco) PHA-P protein/ml. They produced no evidence that this concentration of PHA-P was indeed a stimulus to human lymphocyte proliferation. We and others (16,17) have found that PHA in high concentrations, such as those used by Smith et al. are in fact inhibitory of lymphocyte proliferation. In the present experiments we utilized the most stimulator-y concentration of PHA as determined by [‘Clleucine incorporation into lymphocyte protein. This maximal stimulatory concentration was found to be 10 pg of purified PHA or 820 Fg of PHA-M. Considering that PHA-P is approximately loo-fold more active than PHA-M, it is apparent that the doses of PHA used by Smith et al. were many times greater than those that we used. The present experimental results are not artifactual, because, at various times after establishment of lymphocyte cultures, PGE,, and caffeine, compounds which are known respectively to stimulate adenylate cyclase activity and inhibit phosphodiesterase activity in mammalian cells, produced marked and significant increases in lymphocyte CAMP concentration. In the same experiments PHA in stimulatory concentration failed to induce this effect. Moreover, stimulatory concentrations of commercial PHA and similar concentrations of purified PHA failed to induce an increase in adenylate cyclase activity in lymphocyte membrane preparations. The present results clearly indicate that the stimulation of human lymphocytes by PHA does not involve activation of lymphocyte adenylate cyclase or an increase in intracellular CAMP concentration. In fact, the reverse seems, in general, to be true. For example, we have shown that lymphocyte stimulation is inhibited by PGE1, an agent that causes a prompt and significant rise in intracellular CAMP concentration and in membrane adenylate cyclase activity. This is in agreement with the findings of a number of other groups (4) investigating lymphocyte stimulation in vitro and is also in agreement with the finding of Henney and his associates (6) that mouse lymphocyte activation, as expressed by cytolysis of target cells, was inhibited by adenylate cyclase stimulating agents and by phosphodiesterase inhibitors. We also found that in the presence of the phosphodiesterase inhibitor, caffeine, PHA actually lowered CAMP levels relative to cells treated with caffeine alone. If, as appears likely, lymphocyte activation is accompanied by a fall in the intracellular concentration of CAMP, lymphocytes would conform to what others report for mammalian cells in tissue culture; namely, CAMP levels are lower in dividing or transformed cells than in normal resting cells (18-20).

362

GLASGOW

ET

AL.

ACKNOWLEDGMENTS This study was supported by grants from PHS, NIH and from the American Cancer Society, ET-35B.

CA 10995, AM-10824,

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5. 6. 7. 8. 9.

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STEINER, A. L.. KIPNIS, 1). M., IJTIGEH, R., AND PAHKEH, C., PTOL..Nat. Acud. Sci. 64, 1969. POLGAR, P., VEn.4. C., ICELLEY. P. H., ANI) RUTENBLUG A. M.. Biochem. l3iophy.s. Acts 378, 1973. HUNTER, W. M., AND GREENWOOIX F. C., Nutrrre (Lontfon) 194, 495, 1962. LOWRY, D. H., ROSENBROIJGJ& N. J.. FARK, A. L., 4~11 HANDAI.L. R. J.,./. Riol. C&vi. 265, 1951. WILSON, D. B., J. Exp. Zool. 162, 161, 1966. LING, N. R., In “Lymphocyte Stimulation,” p. 19, Wiley, New York, 1968. SHEPPARD, J. N., Proc. Nut. Acad. Sci. 68, 1316, 1971. SHEPPARI). J. R.. Nature New Biol. 236, 14, 1972. WILLINGHAM, M. C., JOANSON, G. S., AND PASTAN, 1.. Biochern. Hiophys. Re.c. (:omm. 743, 1972.

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