Quantitation of contact-feeding between somatic cells in culture

Primed in Sweden Copvrighr @I 1975 by Academic Press. Inc. AI/ ri,qhrs of reproducmn in an~f<>rrn resrrved

Experimental

QUANTITATION

OF SOMATIC

CHERYL

95 (1975) 3946

Cell Research

CONTACT-FEEDING CELLS

IN

CULTURE

M. CORSARO’ and BARBARA

Department of Pediatrics, Johns Hopkins and Department of Biology, Johns

BETWEEN

University School Hopkins University,

R. MIGEON of Medicine, Baltimore,

Baltimore, MD 21218,

MD 21205, USA

SUMMARY In search for genetic variation among strains of human fibroblasts, we have developed a quantitative assay for contact-feeding between HGPRT+ and HGPRT- cells. The assav. based on the recovery-of 6-thioguanine-re&tant clones from mixtures of these cells, reflects the transfer of 6-thioguanylic acid from wild-type to mutant cells. Results indicate that contact-feeding is cell density dependent and a stable characteristic of adult human fibroblasts. No differences in the degree of contact-feeding were noted among 9 strains of Lesch-Nyhan tibroblast recipients, while skin tibroblasts from 17 adults and 9 children, either normal or with various genetic diseases, did not differ in their ability to act as donors. Using the contact-feeding assay as a potential indicator of mutations involving membrane functions, we noted no differences when tibroblasts from a child with Menkes syndrome and two children with cystic fibrosis were used as donor cells. However, when the human donor cell was of fetal origin, a small, but significant decrease in contact-feeding was noted. When this assay was carried out in the presence of cytochalasin B (0.5-0.7 pg/ml), a small decrease in contact-feeding resulted. The same effect also was observed in autoradiographs of cells exposed simultaneously to cytochalasin B (2 pg/ml) and 3H-hypoxanthine. The results of our studies of contact-feeding between Chinese hamster ovary cells and human tibroblasts are compatible with the hypothesis that gap junctions are the ultrastructural basis for this kind of intercellular communication between cells in vitro [I] and suggest that the extent of communication may be determined by the cell which forms the least number of gap junctions.

The transfer of gene product between cells in contact was first noted in mixtures of normal hamster cells and those with hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency [2, 31. Studies of this kind of cell-to-cell communication in vitro indicate that cells vary in their ability to participate in the process, that cells of one species may communicate with those of another, and that the substance transferred is the nucleotide product of reactions involving HGPRT, adenine phosphoribosyl transferase (APRT), and thymidine kinase (TK) [4,5]. Subak-Sharpe and colleagues referred to I Present address: Department of Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

transfer of gene products by cell contact as ‘metabolic cooperation’ [2]. However, this term might equally apply to transfer of ‘correction factors’ via medium from normal to abnormal cells, as seen, for instance, with mucopolysaccharide disorders [6] and historically referred to as crossfeeding. To distinguish the form of metabolic cooperation mediated by cell contact from cross-feeding, we prefer to use the more precise term ‘contact-feeding’ for the former. This transfer of gene product from normal to mutant cells masks the genotype of the latter and not only interferes with isolation of hybrids from two human fibroblast strains [7], but also impedes the search Expfl

Cd

Res 95 (1975)

40

Corsaro

and Migeon

200 2.5

x104

200 5 x lo4

2Lw IO

5

Fig. I. Contact-feeding assay based on number of clones recoverable from mixed populations of HGPRT+ donor and HGPRT- recipient cells in 6TG

medium. The number of 6TG’ clones obtained from these mixtures is compared with that in the control dish.

for rare mutants among populations where normal human fibroblasts predominate [8111. Therefore, it seemed useful to look for either (1) human fibroblasts which participate in contact-feeding less well than others or (2) methods to inhibit the contact-feeding process. Previous attempts to quantitate the extent of contact-feeding have involved autoradiography, using intensity of label as a measure of product transferred. We report a quantitative assay which permitted comparison of contact-feeding between cells of various human strains and evaluation of the effects of various factors which may influence the process. The assay is based on two sets of observations. The first is that normal cells (HGPRT+) cannot proliferate in the presence of 6-thioguanine (6TG), a substrate for HGPRT. On the other hand, cells lacking HGPRT cannot incorporate this toxic purine analogue and therefore can proliferate in 6TG medium [12, 131. The other observation is that the recovery of HGPRTdeficient cells from artificial mixtures of HGPRT- and HGPRT+ cells decreases as the number of wild-type cells in the mixture increases [8-l I].

We have used this assay, which measures the transfer of 6-thioguanylic acid from wild-type to mutant cells, to show that (1) there is little variation in the degree of contact-feeding among cells of different human strains; (2) the assay may be useful as an indicator of mutations involved in membrane function; (3) cytochalasin B can be inhibitory to a degree, as well as to test the hypothesis that gap junctions are the basis for this kind of intercellular communication.

Exptl

Cell

Res 95 (1975)

MATERIALS

AND METHODS C4S

HGPRT+ cells (donors) Human posfnatal. The HGPRT+ cells strains were from the following individuals: 14 normal adults, 20-57 years of age; 2 normal children, 9-12 years of age; 1 adult with glucose&phosphate dehydrogenase deticiencv: 1 adult with androaen insensitivitv: 1 adult with phosphorylase kinase B related glycogen-storage disease; 2 children with the cri-duchat svndrome: 1 infant with Menkes kinky hair syndrome: 2 children with cystic fibrosis; and 1 child with galactosemia. All of the above were. skin fibroblasts isolated in our laboratory. These cells were used in their third to ninth subculture. Some previously had been frozen in liquid nitrogen in their second or third subculture. Another aalactosemic cell line (GM 54) was obtained from the &tstitute for Medical Research, Camden, NJ, and was used in its eighth subculture. Cells from adult and child donors are r;ferred to collectively as ‘adult’ tibroblasts to distinguish them from cells of fetal origin. <

,

_

I

Contact-feeding

in somatic cells in culture

41

Fig. 2. Abscissa; no. (x 104) of donor (HGPRT+) cells; ordinate: 6TG’ clones (% of control). This figure shows a summary of results for all the assays involving adult human tibroblasts as well as the method of estimating MLD for each donor-recipient pair. To obtain each point on the curve: (1) the number of 6TGr clones recovered (expressed as percent of control) as a function of the number of HGPRT+ donor cells was determined for each of 48 donor-recipient

pairs. (2) These values for a given number of donor cells (i.e., 1~10~) were avenged and the mean and SD. plotted. The dotted line from curve to abscissa indicates the number of donor cells necessary to reduce survival of 6TG’ clones to 50% of control. This value (X 10m4)is the mean lethal dose (MLD). The less the contact-feeding, the greater the number of donor cells needed to reduce survival of 6TG’ clones to 50 % and the greater the MLD .

Feral. The fetal cells were cultured in our labotatory from tissues of 11 non-viable abortuses (50-175 days menstrual age) which had been obtained for another study. Cells in the second to sixth subculture were used from 15 different tissues including lung, liver, heart, skeletal muscle, testes, adrenal, skin, and amniotic fluid. Animal. An HGPRT+ Chinese hamster ovary cell line, CHO-Kl (CCL 61), and the mouse cell line, L-929 (CCL l), were obtained from the American Type Culture Collection, Rockville, MD.

assay was growth medium containing 6x 10m5 M 6-thioguanine (2-amino-6 mercaptopurine, Calbiothem) and is hereafter referred to as 6TG medium. For studies of the effect of serum on contact-feeding, the serum concentration of the 6TG medium was varied from5to20%. Cytochalasin B was obtained from Imperial Chemical Industries, Macclesfield, Cheshire, UK; and dimethyls&oxide (DMSO) was used as a diluent.

HGPRT-

cells (recipients)

The HGPRT- cells were skin fibroblasts isolated in our laboratory from 9 different boys with the Lesch-Nyhan syndrome and were comparable to the donor cells in regard to in vitro age. These cells are resistant to the toxic effects of 6 x 1O-5 M 6-thioguanine (6TG) and clones were obtained in this medium with an efficiency of lO-30%.

Human.

Animal. 6-Thioguanine resistant (6TG’) clones were isolated in our laboratory as spontaneous mutants from Chinese hamster ovary cells CHO-Kl . Using an adaptation of the method of Der Kaloustian et al. [14], these cells were shown to lack HGPRT activity.

Media Cells were maintained in growth medium [Eagle’s minimal essential medium supplemented with nonessential amino acids, glutamine, penicillin-streptomycin, fungizone, and 20% fetal calf serum (GIBCo)] in 60 mm Lux plastic Petri dishes. Medium used for the standard contact-feeding

Contact-feeding

assay

The HGPRT’ cells and HGPRT cells were cocultivated in 6TG medium as follows: A series of 60 mm plastic Petris were preincubated with 5 cc of 6TG medium at 37°C in 5% CO*. HGPRT- cells were trypsinized, suspended in growth medium, counted in a hemocytometer, diluted to 2x lo3 cells/ml of medium, and aliquots of 200 cells were distributed into each dish. Increasing numbers of HGPRT+ donor cells (0, 5X 103, 104, 2.5 X lo’, 5 X 104, 1W) were immediately added to the above dishes. The Petris were reincubated, and medium was renewed the following day and twice weekly. Twelve to 14 days after the cells were plated, the Petri dishes were rinsed twice in 0.85% NaCl, treated 10 min in 95% ethanol, and allowed to air dry. They were stained with toluidine blue and scored for macroscopic clones. The numbers of 6TG’ clones recovered from the mixed cultutes were compared to those recovered when 200 HGPRT- cells had been plated alone, and the result is expressed as a percent of the number of 6TG’ clones in this control dish (fu. 1). The results from each donor-recipient pair were plotted to form a survival curve, as shown in fa. 2. The mean lethal dose (MLD); that is, the number of Erprl

Cell

Res 95 (1975)

42

Corsaro

and Migeotl

HGPRT+ cells (X IO-“) required to prevent the survival of 50% of the 6TG’ clones, was extrapolated from the survival curve. The MLD varies inversely with the ability of a cell to contact-feed. Therefore, the higher the MLD, the less contact-feeding there is. The MLDs for different donor-recipient combinations were then compared and analysed statistically

Table 1. MLD fur assays comparing two strains of recipient cells with a variety of donor strains Donor strains (HGPRT+)

Autoradiography Cells were plated onto 22 mm2 coverslips in 35 mm Petri dishes. Twenty-four hours later, the medium was replaced with I ml of medium containing 3Hhypoxanthine (11.2 Ci/mM, IO @Zi/ml). After 6 h of incubation at 37°C and 5% CO,, coverslips were removed, rinsed in 0.85 % NaCl, and fixed in 95 % ethanol for 10 min. After treatment with 10 % TCA, the coverslips were coated with Kodak NTB2 emulsion, exposed for 8 days, developed, and stained with toluidine blue.

Recipient strains (HGPRT-) no. 12

no. 86

Adult no. 106 “0. 109 no. 103 no. 103

1.6 0.9 1.4 2.1

1.0 I.0 1.3 1.6

Fetal no. no. no. no.

I .4 1.5 2.6 2.7

I.1 1.3 I.5 1.8

K12 K19 K19 K19

RESULTS Our results indicate that the recovery of 6TG’ clones from mixed populations of HGPRT+ and HGPRTcells can be used as a quantitative assay for contact-feeding (fig. 1). When a series of donor strains were tested against each of two recipient LeschNyhan strains, the results did not differ more than repeat determinations for a donor-recipient pair (table 1). These observations indicated that MLD values derived from different strains of LeschNyhan cells might be pooled. Therefore, MLDs for 48 pairs of adult donorlleschNyhan recipient combinations (involving 9 Lesch-Nyhan and 26 HGPRT+ fibroblast strains) were compared to look for variability of contact-feeding among human fibroblasts (fig. 2 and table 2). (Because there were no significant differences between MLDs for child (1.55kO.6) and adult (1.47f0.6) fibroblasts, the results have been pooled. Therefore, the term ‘adult’ refers to cells of both children and adults.) The average MLD for adult skin fibroblast donor and Lesch-Nyhan recipient cells was 1.49kO.56 S.D.; that is, 1.49~10~ donor cells inhibit the proliferation of 50% of Lesch-Nyhan cells. The variance was Exptl

Cell Res 95 (1975)

large, but not attributable to obvious differences in either donor or recipient cells. Differences in MLD were not related to sex of the donor (table 2), nor to in vitro age of either donor or recipient cells. Moreover, the variation was similar to that observed between repeat determinations for the same donor-recipient combinations (table 1). As seen in table 2, the average MLD for fetal donors and Lesch-Nyhan recipient combinations was 2.56+ 1.25 S.D., which is significantly different (p
Contact-feeding

in somatic cells in culture

Table 2. Extent of contact-feeding

as measured by MLDa

Recipient cells (HGPRT-)

Donor cells (HGPRT+)

No. of donor strains tested

Average MLD+S.D.

Human b

Human adult Female Male Human fetal Hamster (CHO) Hamster (CHO) Human adult Mouse (I,) Menkes disease Cystic fibrosis

48

1.49f0.56 1.45*0.51 1.54+0.63 2.56k1.25 28.00 30.80 21.50 >50.00 2.70 1.75

Human Hamster (CHO)‘. Human Hamster (CHO) Human Human Human

:: 37 2 2 2 1 :

LI Note MLD refers to the number of donor cells (X 10m4) required to prevent the proliferation clones; therefore, the higher the MLD, the less the contact-feeding. * Human HGPRT- cells were from 9 unrelated Lesch-Nyhan males. ’ Hamster HGPRT- cells were from 6TGr clones of CHO cells.

mm, each filled with 2 ml of medium. Thus, the dishes varied in the number of cells/ mm2. The MLDs in the 35 and 60 mm dishes were 0.7 and 1.4, respectively. Contactfeeding was proportional to the cell density, confirming previous reports that cell contact is required for contact-feeding to occur [3,5, 181. Efforts were made to decrease the extent of contact-feeding between cells in culture by varying the serum concentration in the medium. The amount of contact-feeding decreases as the concentration of serum in the medium is lowered from 20 to 5 %, as reflected by the*increase in MLD (table 4). We also explored the possibility that cytochalasin B, a compound influencing some membrane functions, such as the transport Table 3. MLDs for assays involving combinations of Chinese hamster (CHO) and human cells HGPRT+ donoF.. CHO Human CHO Human HGPRT- recipienta . .. CHO CHO Human Human Expt 1 Expt 2

36.0 28.0 20.0 15.0

45.0 16.5

2.1 1.6

a Donors were either CHO-KI or human adult fibroblast strain. b Recipients were either a STG-resistant clone of CHO-KI or Lesch-Nyhan fibroblast strain.

43

of 50 % 6TG’

of glucose [IS] and nucleosides [16], might affect communication between cells. This compound, an inhibitor of cytokinesis, has been shown to alter cell morphology [17]. Therefore, the dose of cytochalasin B used, while permitting cytokinesis essential to the contact-feeding assay, did change the shape of the fibroblasts towards that of an epithelial cell. The MLD for cells exposed to cytochalasin B is greater than for cells exposed to the diluent alone, indicating some effect leading to a decrease in contact-feeding (table 5 a). The change in MLD, however, is not great and may be influenced by the culture conditions since cells showed some toxicity in the presence of cytochalasin B. For this reason, we looked at the effect of this drug on contact-feeding as determined by autoradiography. Table 5 b presents the results of the autoradiographic studies. The numbers of labeled and unlabeled cells (scored on the basis of intensity of label as 0, +, and +++) in 1: 1 mixtures of ++, HGPRT+ and HGPRTcells have been compared with those of each kind of cell in pure culture. HGPRT+ cells in pure culture have either ++ or + ++ labeling, while HGPRTcells uniformly have 0 laExptl

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Res 95 (1975)

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Table

4. Effect of serum concentration upon contact-feeding: MLD for assays carried out at various serum concentrations Concentration of fetal calf serum Expt

5%

10%

15%

20%

no. 1 no. 2 no. 3 Average MLD SD.

3.9 3.7 2.6 3.4 +0.7

3.4 2.8 2.8 3.0 f0.4

1.9 2.2 2.3 2.1 +0.2

1.6 2.3 1.7 1.9 f0.4

beling. Therefore, the cells with intermediate levels of labeling (+), found only in the mixed population, must represent HGPRTcells which have been contactfed by HGPRT+ donor cells. In the presence of cytochalasin B, there are significantly more cells with 0 labeling and significantly less cells with + labeling than in growth medium @
This study was undertaken to search for genetic variation among human cells with respect to contact-feeding. Since there are mammalian cells (such as L cells) which do not readily contact-feed [5] or form gap junctions [ 11, it was hoped that a similar human variant could be found to use for cell selection studies. We determined the MLD for cells like L and CHO-K 1, which participate poorly in contact-feeding (see table 2), and used these values as yardsticks to evaluate MLDs for a variety of human cell Erptl

Cell Res 95 (1975)

strains. By this standard, we did not observe any human cells unable to contactfeed. In fact, our results indicate that degree of contact-feeding is remarkably uniform for all the human cells assayed. The only statistically significant variation found was that between adult and fetal cells. This may reflect differences between fetal and non-fetal membranes resulting in fewer sites on the surface of the fetal cell for the formation of gap junctions. On the other hand, more trivial explanations might apply. The fetal cell may have a smaller surface area, and thus more fetal than adult cells would be necessary to reach the same effective level of contact-feeding. Another factor might be the relative heterogeneity of the fetal cell populations analysed. In contrast to adult specimens, which were of dermal origin and consisted entirely of fibroblasts, the fetal specimens were derived from a variety of tissues; and many specimens included epithelial cells as well as fibroblasts. Although no single type of fetal tissue significantly differed from another, the heterogeneity of cell type within a tissue might account for the larger variance observed in fetal cells than in adult cells. The proportion of epithelial cells in each specimen could not be determined. Attempts to compare contact-feeding by epithelial versus fibroblast donors were unsuccessful because many of the former cells did not attach to the Petri dish. Table 5a. Effect of cytochalasin upon contact-feeding as determined by recovery of 6TG-resistant clones MLD Dose cytochalasin bidmU

Cytochalasin

DMSO control

0.5 0.7 0.7

4.4 5.4 1.6

2.3 2.0 1.3

Contact-feeding Table 5 b. Effect of cytochalasin

upon contact-feeding % Labeled cells”

Medium Growth*

Cytochalasin (2 f.dml)

Cells exposed to 3H-hypoxanthine

0

HGPRT+ HGPRT1 : 1 mixture

1 100 13

HGPRT+ HGPRT1 : 1 mixture

1 100 28

in somatic cells in culture

as determined

45

by autoradiography

+

++

+++

No. of cells scored

i 48

79 0 31

15 0 8

799 701 756

2 0 31

84 0 29

13 0 12

565 525 627

o 0, no label over background; +, scant label; + +, nucleus well labeled; + + +, nucleus heavily labeled. b Growth medium contains same amount of DMSO diluent (0.07 %) as does medium containing cytochalasin B.

Because of the stability of the contactfeeding phenotype among normal human cells, the assay could be used as a means of detecting variants among cells suspected of having mutations involving transport or other membrane functions. We tested donor cells from individuals with cystic fibrosis and Menkes syndrome, a disease related to a mutation resulting in defective copper transport, but found no differences from wild-type donors in their ability to contactfeed. Factors which injluence extent ofcontact-feeding We have considered the CHO-Kl cell as a model for a cell which participates poorly in the transfer of gene product from one cell to another. It has been postulated that cells which contact-feed poorly have relatively few gap junctions, thus limiting the transfer or receipt of cell products. In fact, Gilula et al. [l] have suggested that gap junctions are the ultrastructural basis for this kind of communication between cells. The suggestion was based on observations using electron microscopy and freeze-etching techniques that both contacr-feeding and ionic coupling in fibroblasts are associated with the presence of gap jenctions. As a test of this hypothesis, we com-

pared MLDs from various Chinese hamster-human cell combinations. The results show that the presence of the CHO-Kl cell as either donor or recipient severely inhibits contact-feeding to the same degree observed when both participants are CHOKl cells. Therefore, these results not only are compatible with the theory that membrane sites such as gap junctions are the ultrastructural basis for contact-feeding, but suggest that the deficiency of these junctions-in either donor or recipient-is equally able to inhibit the transfer of product from one cell to the other. Thus, the extent of contact-feeding is determined by the participant which forms the least number of gap junctions. These results would be equally compatible with other models; for example, deficiency of other membrane sites or pertinent receptors active in efferent as well as afferent processes. Our results, using the contact-feeding assay, indicate that cell density is critical to the process as suggested previously by others on the basis of autoradiography [3, 5, IS]. Furthermore, we observed that contactfeeding (as defined by MLD) decreases as the serum concentration in the medium decreases. This may indicate the existence of serum factors facilitating the formation Exptl

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Res 95 (1975)

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of gap junctions or the passage of molecules between these junctions. However, it may simply be that cells adhere better to the surface of the Petri dish in higher concentrations of serum thus enabling more cell contacts to be made. Microscopic inspection of the Petris after several days in culture did in fact reveal more unattached cells in Petris with a lower serum concentration than in those with a higher serum concentration. Cytochalasin B has been shown to inhibit microfilament function [ 191 and to decrease the transport of glucose [15] and nucleosides [16] into cells and so might be expected to affect the transfer of gene products from cell to cell. This was tested in two ways. When the contact-feeding assay was carried out in the presence of cytochalasin B, a slight increase in the recovery of 6TGresistant clones was noted, indicating some decrease in cell communication. The second method, using autoradiography to detect the passage of labeled nucleotide from one cell to another, enabled higher doses of the drug to be used since cytokinesis is not required for this assay. In the presence of cytochalasin B there were fewer cells with intermediate labeling than in growth medium, thus fewer HGPRT- cells were contact-fed by HGPRT+ cells. These results were consistent with recent autoradiographic observations that cytochalasin B can inhibit contact-feeding [20]. The cytochalasin B effect may well be attributable to the changes it induces in fibroblast morphology. We observed that treated fibroblasts had fewer of the processes characteristic of human fibroblasts and assumed an epithelioid morphology. Without processes, a cell is less apt to make contact with another cell. The possibility exists, however, that the cytochalasin B effect is due to an actual physical change ExprlCellRes

95 (1975)

in the cell membrane interfering with gap junction formation. Nonetheless, by standards of CHO and L cell responses, the effect is a small one; thus cytochalasin B, in doses which do not inhibit cytokinesis, is not effective in blocking this intercellular communication to any significant degree. Although our studies did not reveal any common variant among human cells, they did indicate the value of a quantitative assay for the study of intercellular communication in vitro. We are grateful to Dr Gary Chase for discussions related to statistical analysis of the data. This investigation was supported by NIH research grant HD 05465. C. M. C. is a pre-doctoral fellow supported by NIH training grant GM-57.

REFERENCES 1. Gilula, N B, Reeves, 0 R & Steinbach, A, Nature 235 (1972) 262. 2. Subak-Sharpe, H, Btirk, R R & Pitts, J D, Hereditv 21 (1966) 342. 3. l J cell sci 4 (1969) 353. 4. Cox, R P, Krauss, M R, Balis, M E & Dancis, J, Proc natl acad sci US 67 (1970) 1573. 5. - EXD cell res 74 (1972) 251. 6. Neufeid, E F & Cantz, M J, Ann NY acad sci 179 (1971) 580. 7. Migeon, B R, Norum, R A & Corsaro, C M, Proc natl acad sci US 71 (1974) 937. 8. Albertini, R J & DeMars, R, Science 169 (1970) 482. 9. Migeon, B R, Nature 239 (1972) 87. 10. VanZeeland, A A, van Diggelen, M C E & Simons, J W I M, Mut res 14 (1972) 355. 11. Stark, R M & Littlefield, J W, Mut res 22 (1974) 281. 12. Seegmiller, J E, Rosenbloom, F M & Kelley, W N, Science 155 (1967) 1682. 13. Migeon, B R, Biochem genet 4 (1970) 377. 14. DerKaloustian, V M, Byrne, R, Young, W J & Childs, B, Biochem genet 3 (1969) 299. 15. Estensen. R D & Plaeemann. P G W. Proc natl acad sci US 69 (1972) r430. 16. Planemann. P G W & Estensen. R D, J cell biol55 (1972) 179.’ 17. Puck, T T, Waldren, C A & Hsie, A W, Proc natl acad sci US 69 (1972) 1943. 18. Dancis, J, Cox, R R, Berman, P, Jensen, V & Balis, M E, Biochem genet 3 (1969) 609. 19. Wessels, N K, Spooner, B S, Ach, J F, Bradley, M 0, Luduena, M A, Taylor, E L, Wrenn, J T 8t Yamada, T C M, Science 171 (1971) 135. 20. Cox, R P, Krauss, M R, Balis, M E & Dancis, J, J cell physio184 (1974) 237. Received February 28, 1975