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J. Cell Sci. 7, 671-682 (1970) 67!

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GROWTH AND MACROMOLECULAR

SYNTHESIS IN THE 3T6 MOUSE FIBROBLASTI. GENERAL DESCRIPTION AND THE ROLE OF

ASCORBIC ACID

C. I. LEVENE AND C. J. BATESDunn Nutritional Laboratory, University of Cambridgeand Medical Research Council, Cambridge, England

SUMMARY

The growth of the j T6 mouse fibroblast and synthesis of macromolecules has been studiedover the last 6 days of the cultures' 14-day life span. The effect of ascorbic acid was also tested.

During this period, the cells synthesized collagen, as judged by the appearance of non-dialysable hydroxyproline, which was identified by chemical assay and by radioactive incor-poration studies. A high proportion of the collagen in the cell layer was insoluble ino-iN aceticacid. Of the hydroxyproline synthesized in the presence of ascorbic acid, about 75 % waseventually liberated into the growth medium, and about 25-30% of the liberated materialbehaved as free hydroxyproline. In the absence of ascorbic acid, the cell layer hydroxyprolinewas reduced to one-third, but the growth medium hydroxyproline was unaffected.

The cells also synthesized glycosaminoglycans, as judged by the appearance of cetyl pyri-dinium-precipitable uronic acid, and the incorporation of labelled glucosamine into macro-molecules. A large proportion of this material has the properties of hyaluronic acid. Ascorbicacid had no detectable effect on overall glycosaminoglycan synthesis, in contrast to healingtendonectomy wounds in guinea-pigs.

Cell proliferation and general protein synthesis were virtually unaffected by ascorbic acid.Whereas general protein synthesis, like cell proliferation, declined in the ageing culture,glycosaminoglycan synthesis and collagen synthesis continued at a steady or increasing rate.

INTRODUCTION

The fibroblast synthesizes collagen and glycosaminoglycans (GAG's), and it hasrecently become possible to develop stable lines from this cell type which grow intissue culture for many generations and still retain both of these typical connectivetissue cell properties (Green & Todaro, 1967). Ascorbic acid has a clear-cut role inthe biosynthesis of collagen (Udenfriend, 1966; Prockop & Kivirikko, 1967; Barnes,1969), but its role in the biosynthesis of GAG's, or in the organization of connectivetissue elements, remains to be defined (Bates & Levene, 1969). The purpose of thisstudy is to explore the relationship between cell growth and synthesis of the macro-molecular components of fibrous tissue.

The cell line used, 3 T6, was chosen because its derivation, collagen-synthesizingability and dependence on added ascorbic acid have already been described in somedetail, mainly by Green and co-workers (Todaro & Green, 1963; Goldberg, Green &Todaro, 1963; Green & Goldberg, 1963, 1964; Priest & Bublitz, 1967).

This paper describes some observations on the growth and maintenance of j T6,its ability to synthesize macromolecules, and the effect of ascorbic acid.

672 C. I. Levene and C. J. Bates

MATERIALS AND METHODS

Growth and harvesting of cultures

Green's 3 T6 mouse fibroblasts were obtained from Dr Mary Daniels of the StrangewaysResearch Laboratory, Cambridge, England. They were grown, fed and subcultured as describedby Todaro & Green (1963), with the exception that the concentration of glucose was halvedand the medium was equilibrated with 5 % CO2 instead of 10%, the concentration of bicar-bonate being therefore reduced by half as recommended by Kochhar, Dingle & Lucy (1968).Cells plated at 25000/60-mm plate survived 14-15 days before curling up, and ascorbic aciddeprivation does not appear to prolong survival of the cultures. When curling occurred, it wasimmediately accompanied by a drastic reduction in macromolecular synthesis and the experi-ments were therefore always terminated before the cell layers curled. Since the cells grew

— Ascorbace + / — Ascorbate

14C-ProAdd < 14C-Trp

"C-Glu

S e e d

25000cells/dish

Harvest

0 6 8 10 12 14Culture age, days

Fig. 1. Design of isotope incorporation and macromolecular synthesis experimentsin crowded cultures. 14C-Pro: L-[U-14C]proline, 0-05 /tCi/plate. 14C-Trp: methylene-L-[14C]tryptophan, o-io/tCi/plate. 14C-Glu: D-[i-14C]glucosamine, C05/tCi/plate.

logarithmically for the first 8-10 days the period of synthesis in a fairly static culture waslimited to the last 4-6 days. Throughout the 14-15 days of culture, the cells were fed 3 timesa week with fresh growth medium, and ascorbic acid (50 /tg/ml) was included on and afterthe 8th day (Fig. 1). Inclusion at the time of plating adversely affected the proportion of cellswhich adhered to the plastic.

For cell numbers and DNA determinations the cell layers in triplicate were trypsinized andcounted in a haemocytometer or in a Coulter Counter, model B (Coulter Electronics, Hialeah,Florida). The remainder of the cells were spun down into a pellet and used for DNA analysis.

Older cultures were treated with collagenase (Worthington CLSPA) before trypsinization.

Chemical analyses

DNA was measured by the method of Burton (1956).Collagen synthesis. Collagen in the cell layer was measured by the method of Bergman &

Loxley (1963) (rapid procedure A), or by the method of Prockop & Udenfriend (i960) afterhydrolysis at 100 °C for 24 h in 6N HC1. The second method was used for estimations in thegrowth medium. Certain hydroxyproline measurements were checked by analysis on theTechnicon amino acid autoanalyser (Technicon Instruments Co. Ltd., Hanworth Lane,Chertsey, Surrey). Undialysed serum was found to contain 25 /tg/ml hydroxyproline; the con-tribution per plate (10% serum, i.e. 0-4 ml serum per dish) was therefore 10 fig. Dialysedserum (04 ml per dish) contributed only 2 /ig per dish. In calculating the quantities liberated

Synthesis in jibroblasts and role of ascorbate 673

by the culture into the growth medium, blank values obtained with unused medium weretherefore routinely subtracted. Good agreement was then obtained between the Prockop &Udenfriend and the Technicon measurements.

The solubility characteristics of the cell layer collagen were measured in the followingmanner. 20 /tCi of L-[G-3H]proline (266 mCi/mmol) were added to 10-day-old cultures in2 feeds, and the cultures were harvested 4 days after the first radioactive dose. Salt-solublecollagen was extracted from several pooled cell layers by shaking at 4 °C in 5 volumes of1 M NaCl, buffered at pH 7 6 with o-oi M sodium phosphate, for 8 h. Acid-soluble collagen wasthen extracted by shaking in 5 volumes of O I N acetic acid for 16 h, and the remaining materialwas designated 'insoluble'. The amount of hydroxyproline in each fraction was measured afterhydrolysis, by the method of Bergman & Loxley (1963), and its radioactivity content wasassayed by the method of Juva & Prockop (1966).

GAG synthesis. Uronic acid was estimated by the Galambos (1967) modification of theDische uronic acid assay in the cell layer and the growth medium after papain digestion andseparation of the GAG's present by cetyl pyridinium precipitation (Schiller, Slover & Dorfman,1961). Judging from spectra obtained in this assay, material prepared from the growth mediumwas uncontaminated but that prepared from the cell layer was still contaminated with non-uronic acid, carbazole-positive compounds. In addition, some of the mucopolysaccharidematerial failed to precipitate with cetyl pyridinium chloride. Further experiments indicated thatin the case of the growth medium material, a satisfactory correction factor, in the region of1'5—20 could be derived from the radioactive glucosamine distribution measurements. Thiscorrection could not be applied to the cell layer material however, because of the likely presenceof hexosamine-containing glycoproteins, in addition to the mucopolysaccharides.

Radioactive precursor incorporation. In certain experiments, radioactively labelled precursorswere added to the growth medium, to measure the synthesis of macromolecules over a 2-dayperiod, immediately prior to harvesting the cells (Fig. 1). The labelled substances were:L-[U-l4C]proline (90 mCi/mmol) for measurement of general protein and collagen synthesis;methylene-L-[14C]tryptophan (54/5 mCi/mmol) for measurement of general protein synthesis,and D-[i-14C]glucosamine (405 mCi/mmol) for measurement of mucopolysaccharide syn-thesis. All these labelled precursors were obtained from the Radiochemical Centre, Amersham.The doses routinely given were: glucosamine and proline, 0-05 /tCi/plate; tryptophan, o-io /tCi/plate. After harvesting, the cell layers labelled with proline or tryptophan were washed twicewith 10 ml 10% trichloroacetic acid either containing i-omg/ml L-proline or saturated withDL-tryptophan, respectively, and dissolved in a scintillation mixture containing: toluene, 800ml;Beckman Biosolv BBS-3, 200 ml; and Scintillator Butyl-PBD (Ciba), 8 g. The proline-labelledsamples, after hydrolysis, were assayed for labelled hydroxyproline by the method of Juva &Prockop (1966). The glucosamine-labelled cell layers were subjected to proteolysis and to cetylpyridinium precipitation as described above, and aliquots of the soluble and insoluble fractionswere dissolved in the scintillation mixture described above. Aliquots of the labelled growthmedia were analysed directly for total radioactivity, and again after exhaustive dialysis againstdistilled water containing unlabelled substrate (001 %) . Samples of feed solutions containingthe isotope were similarly treated to check the completeness of free isotope removal. All sampleswere counted in a Packard Tricarb scintillation spectrometer, model 3375.

Characterization of growth medium GAG's. The medium obtained after growth in the presenceof D-[i-14C]glucosamine was dialysed, the diffusible fraction was analysed by gel filtration on acolumn of Sephadex G25, and the non-diffusible fraction was analysed on columns of Sepha-dex G75 and Sepharose 4B. Fractions were analysed for radioactivity, and the peak positionswere compared with those of Blue Dextran and chloride ion markers.

Quantitative expression of the chemical and isotopic data. Of the various possible bases ofcomparison, we have chosen to express the results per unit of io° cells. The cell layer hydroxy-proline and uronic acid values represent amounts accumulated over the whole growth period,whereas the amounts found in the growth medium are accumulated for 2- or 3-day periodsonly, being the interval between successive feeds. The summated growth medium values forthe period considered were compared with the total amount accumulated in the cell layer. Theisotopic data, in contrast, relate only to the 2-day period prior to harvesting, for both cells andgrowth medium. They have therefore been compared directly, without combining measure-ments at the different time points.


RESULTS

Growth

Cells plated at 200000 per plate grew to 3-46 x io6 per plate in 72 h giving a doub-ling time of about 18 h. If plated at 25000 per plate, they grew logarithmically for8-10 days, followed by a stationary phase of 4-6 days, terminated by curling (Fig. 2).

u

Culture age, days

Fig. 2. Growth curve in the presence of ascorbic acid. Cell numbers were measuredin the Coulter Counter, after trypsinization, dispersal, and dilution with Earle'smedium.

Effect of ascorbic acid on maintenance

In the absence of ascorbic acid, the final count, expressed as io6 cells, was 11-34 +°"65%; in its presence, 12-25+0-66 (7 experiments). Ascorbic acid thus increasedthe final cell count by 8-3% ± 3-91 %. 0-4 > P > 0-2.

Hydroxyproline production

Effect of age. Table 1 shows the course of hydroxyproline appearance in the celllayer and growth medium over the last 6 days in the presence of ascorbic acid. Clearlyboth of these compartments increase steeply with time.

From Table 2 it is seen that, of the growth medium hydroxyproline, 25-30% isfree and the remainder bound, calculated from hydroxyproline measurements(Prockop & Udenfriend, i960, method) on hydrolysed and unhydrolysed samples. All

Synthesis in fibroblasts and role of ascorbate 675

3 compartments were analysed separately, and the sum of non-dialysable hydroxy-

proline, plus free hydroxyproline, was found to be equivalent to the total hydroxy-

proline present. All the bound hydroxyproline must therefore be in a high molecular

weight form.

Table 3 gives the corresponding isotopic data for hydroxyproline formation at

Table 1. Cumulative hydroxyproline values for cell layer and

growth medium in the presence of ascorbic acid

Growth medium Cell layerCulture age, hydroxyproline, hydroxyproline,

days /<g/iof' cells /tg/106 cells

81 0

1 2

14

o-8o2-473-85556

0-24

0-49I-6I

2 0 2

Cell layer values are the amounts present in the cell layer at each of the time points shown;growth-medium values are the sum of the amounts liberated during each 2-day collectionperiod, from the eighth day until the time of harvesting at 10, 12 or 14 days.

Table 2. Growth-medium hydroxyproline compartments in the

presence and absence of ascorbic acid in crowded cultures

Hydroxyproline content, /«g/ioG cellsA

f \

— ascorbate + ascorbate Ratio:Compartment (A) (B) A/B

Whole growth medium 3-09 314 099Non-diffusible fraction 2-30 226 102Free hydroxyproline 0-78 0-91 086

The figures shown are cumulative values for 6 days growth. The cell layer hydroxyprolineratio in this experiment, A(— ascorbate)/_6( + ascorbate), was 037.

Table 3. Formation of [MC]hydroxypro line in washed cells and

dialysed growth medium in the presence of ascorbic acid

Culture age,days

81 0

1 2

14

Dialysed growthmedium

['•'CJhydroxy-proline,

dpm/10" cells

76

155889 0

Cell layer[14C]hydroxy-

proline,dpm/106 cells

47iS72062 0 0

The incorporation has occurred during a 2-day period immediately before harvesting at thetime points shown.


2-day intervals for the final 8 days. In this case, data are given for dialysed medium, i.e.

hydroxyproline in macromolecules only, since the values obtained with undialysed

medium tend to be distorted as a result of interference from the large excess of free

proline present. The ratio of cell layer to growth medium hydroxyproline increases

Table 4. Solubility of cell layer collagen in a \\-day

culture grown in the presence of ascorbic acid

Total hydroxyproline Radioactive hydroxyproline

Collagen fraction /tg/plate % of total dpm/plate % of total

Neutral salt-soluble 0-30 15 7700 8Acid-soluble 0-52 27 22700 25Insoluble 117 59 60800 67

Cells were grown for 6 days after the addition of ascorbic acid, with 2 feeds each containing20 /tCi [3H]proline per plate.

Table 5. Comparison of the effects of culture age and ascorbic

acid status on hydroxyproline formation

Culture age,and

ascorbic acid

8 days, minusacid

days,

status

ascorbic

14 days, minus ascorbicacid

14 days, withacid presentlast 6 days

ascorbicfor the

The data

Total hydroxyproline

Growthmedium

//g/ios cells

o-8o

S'79

556

are expressed as

Celllayer

/tg/io* cells

0-24

0-64

2-02

in Tables 1 and

[14C]hydroxyproline

Growthmedium

dpm/100 cells 1

76

79

90

3-

Celllayer

dpm/100 cells

47

9 0

2 0 0

Table 6. Total incorporation of [uC]proline in washed cells

and dialysed growth medium in the presence of ascorbic acid

Culture age,days

Dialysedgrowth medium

[14C]proline,dpm/106 cells

Cell layer[14C]proline,

dpm/106 cells

8IO

1 2

14

9 1 3

9536 1 2

7 1 2

4320

266022102100



with time, and the total incorporation rate reaches a plateau at the time that cellnumbers reach their plateau.

The chemical and isotopic data in Table 4 indicate that 60-70% of the collagen isinsoluble at 14 days.

Effect of ascorbic acid. Table 5 compares the effects of age and ascorbic acid statuson the formation of hydroxyproline (total synthesis and radioactive incorporation). Itis clear that while ascorbic acid has a marked stimulatory effect on cell layer hydroxy-proline, its effect on the growth-medium compartment is negligible. Moreover, acomparison of the free and bound hydroxyproline fractions of the growth mediumfrom ascorbic acid-supplemented and unsupplemented cultures (Table 2) indicatesthat, under the conditions specified, both fractions are unaffected by the vitamin. Incertain experiments, where the cells were harvested at a lower density, ascorbic aciddid have a stimulatory effect on growth medium hydroxyproline, but the stimulationobserved in the cell layer was about 10-fold greater. Since the growth medium repre-sents 75% of the total hydroxyproline formed in the system, ascorbic acid causesonly a 16% increase in overall hydroxyproline production, despite a 2- to 3-foldstimulation in the cell layer.

Non-collagenous proteins

Effect of age. Table 6 shows that, unlike [14C]hydroxyproline formation, the totalincorporation of [14C]proline per cell falls with time, and the ratio of cell layer togrowth medium incorporation does not change with age. A similar pattern wasobserved with [14C]tryptophan (Table 7), which provides confirmatory evidence thatcollagen behaves differently from non-collagenous proteins, under these conditions.

Table 7. Incorporation of [uC]tryptophan in washed cells anddialysed growth medium in the presence of ascorbic acid

Culture age,days

Dialysedgrowth medium[14C]tryptophan,

dpm/106 cells

Cell .layer[14C]tryptophan,

dpm/100 cells

8 1310 768014 1200 4960


Effect of ascorbic acid. As seen in Table 8, ascorbic acid had little or no effect ontotal proline and total tryptophan incorporation in the cell layer or the growth medium.Overall protein synthesis (or turnover) therefore does not appear to be affected byascorbic acid under these conditions.


Table 8. Effect of ascorbic acid on [uC]proline, [uC)tryptophan

and [uC]glucosamine incorporation into macromolecules, and uronic

acid production, at 14 days

Assay

[14C]prolineincorporation

[14C]tryptophanincorporation

[14C]glucosamineincorporation

— ascorbic acidA

Cell layer,dpm/io6 cells

1700

4980

2400

/tg/106 cells

Growth medium,dpm/106 cells

690

1130

2570

/tg/106 cells

+ascorbic acid1

Cell layer,dpm/106 cells

2100

4960

2000

jig/10" cells

Growth medium,dpm/106 cells

7 1 2

1200

2510

/tg/106 cells

Uronic acidproduction

17-0 I3-4 17-2 1 3 9

[14C]proline and [14C]tryptophan: total incorporation into washed cells and dialysed growthmedium.

[14C]glucosamine: total incorporation into protease-soluble, non-dialysable compounds incell layer and growth medium.

Uronic acid production: cumulative values for protease-soluble, cetyl pyridinium-insolublecompounds.

Table 9. Uronic acid formation in cell layer and growth

medium in the presence of ascorbic acid

Culture age,days

81 0

1 2

14

Growth-mediumuronic acid,/tg/io6 cells

6-718-433'3S°-5

Cell-layeruronic acid,/*g/io6 cells

i4-S11-4

I4'3n - 4

Cell layer values are the amounts present in the cell layer at each of the time points shown;growth medium values at each time point are the sum of the amounts liberated during the2-day collection periods, from the eighth day until the time of harvesting at 10, 12 or 14 days.

Production of uronic acid-containing macromolecules and [uC]glucosami?ie incorporation

Effect of age. From Table 9, it can be seen that the amount of CPC-precipitable

uronic-positive material in the cell layer matched the cell count closely throughout the

period considered. As noted in the Methods section, however, the recovery and purity

of recovered material here are in some doubt. The material in the accumulated growth

media rose sharply with time, indicating a continuous output from the cells during the

period considered, but no progressive accumulation in the cell layer.

It is evident from Table 10 that incorporation of [14C]glucosamine in both the cell


layer and the growth medium is fairly constant over the period considered, and the2 compartments show approximately equal incorporation; likewise the CPC-precipi-table fraction of the cell layer material shows only small changes with time.

Effect of ascorbic acid. As seen from Table 8, ascorbic acid had no significant effecton either [14C]glucosamine incorporation or macromolecular uronic acid production,either in the cell layer or in the growth medium compartments. Overall mucopoly-saccharide synthesis (or turnover) therefore does not appear to be affected by ascorbicacid under these conditions.

Table 10. [i*C]glucosamine incorporation into macromolecules in thecell layer and growth medium in the presence of ascorbic acid

Cell layer, [14C]glucosamineDialysed

growth medium, Cetyl pyridinium Cetyl pyridinium-Culture age, [14C]glucosamine, Total, -soluble, insoluble,

days dpm/100 cells dpm/10" cells dpm/10" cells dpm/106 cells

8 3210 2174 1400 774

10 2140 1557 900 657

12 2060 1685 895 790

14 2450 2002 972 1030


Preliminary characterization of [uC]glucosamine-labelled products

The diffusible fraction from untreated growth medium revealed no material largerthan glucosamine. The non-diffusible material was excluded by Sephadex G 75;about 50% appeared as a single peak at the void volume of Sepharose 4B; the re-mainder was spread out, but was nevertheless eluted before albumin. The majority ofthe glucosamine-labelled material in the growth medium is therefore of high molecularweight, and breakdown products characteristic of hyaluronidase activity or lowmolecular weight glycoproteins are apparently not present. Other characteristics ofthis polymer: sensitivity to testicular hyaluronidase, nature of the hexosamine( > 90% glucosamine), and small or negligible sulphate incorporation, all tend to con-firm the current opinion that the major part of it is, or closely resembles, hyaluronicacid.

Similar material of very high molecular weight could be extracted from the celllayer by sonication in water. Of the radioactive material which remained insolubleat this stage (about 50% of the total present in the cell layer) the majority could beliberated into solution by the standard proteolytic treatment with papain and pronase.Of this 82% was non-dialysable.


DISCUSSION

We have examined the synthesis and cross-linking of collagen and the synthesis ofnon-collagenous proteins and mucopolysaccharides by j T6 fibroblasts in the presenceand absence of vitamin C.

Hydroxyproline, a characteristic component of collagen, occurs in 3 major com-partments: high molecular weight material (fibrillar collagen) in the cell layer; highmolecular weight soluble material in the growth medium, and free hydroxyproline inthe growth medium. No significant quantities of small peptides could be detected.Total growth medium hydroxyproline is 2-to 3-fold higher than that in the cell layer,and, unlike the cell layer collagen, both of the growth medium hydroxyproline com-partments are virtually unaffected by the ascorbic acid status of the cultures.

The liberation, by cultured cells, of large amounts of hydroxyproline into thegrowth medium has previously been described by several laboratories: Macek,Hurych & Chvapil, 1967; Schafer, Silverman, Sullivan & Robertson, 1967; Aleo,1969; Priest & Davies, 1969. In one instance (Schafer et al. 1967) it was also noted thatvitamin C affects growth medium hydroxyproline much less than that of the cell layer

Presumably, the effect of vitamin C on cell-layer hydroxyproline arises from itseffect on the synthesis of collagen, since it is a cofactor for the enzyme protocollagenhydroxylase. In contrast, free hydroxyproline, which probably arises by degradation,is likely to be unaffected by ascorbic acid (Grillo & Gross, 1967). Preliminaryexperiments by J. Steinberg (personal communication) suggest that newly labelledhydroxyproline in the cell layer of 3 T6 is rapidly liberated into the growth mediumand that the rate of liberation is independent of ascorbic acid. The existence of adelicate balance between collagen synthesis and degradation has been reviewed byWoessner (1968) and was clearly illustrated in tissue cultures of bone by Stern,Glimcher & Goldhaber (1966).

However, it is more difficult to account for the existence of the high molecularweight growth-medium compartment, and for its apparent independence of ascorbicacid. If it is a precursor of cell-layer collagen, a further ascorbic acid-dependent step ispresumably needed for the conversion. Other possibilities are that it represents partialdegradation of newly synthesized collagen, or abortive or incomplete synthesis. Furtherstudies are in progress to determine the nature and ultimate fate of this material.

Like Schafer et al. (1967), we find that once the newly synthesized collagen is de-posited in the cell layer, it rapidly becomes insoluble in neutral salt and acetic acidsolutions. It should therefore provide a useful model for the study of cross-linkingprocesses in a controlled environment, and studies with lathyrogens and otherinhibitors of cross-linking are in progress.

The major mucopolysaccharide produced by j T6 under our growth conditionshas the properties of hyaluronic acid, and does not contain galactosamine. It is pro-duced throughout the growth cycle and its synthesis, like that of collagen, remainshigh when cell division and general protein synthesis decline, during the stationaryphase of growth. In human dermal fibroblasts in tissue culture (Schafer et al. 1968)and in healing tendonectomy wounds in guinea-pigs (Bates, Levene & Kodicek, 1969),


the synthesis of certain galactosamine-containing sulphated mucopolysaccharides isinfluenced by ascorbic acid. It will therefore be of interest to determine whether j T6synthesizes detectable quantities of such compounds and whether they are ascorbicacid-dependent.

We are indebted to Dr E. Kodicek for helpful discussion, Mr B. J. Constable and Mr L. F.Morton for analyses of hydroxyproline on the Technicon autoanalyser, and to Mr B. Pope fortechnical assistance.

REFERENCES

ALEO, J. J. (1969). Collagen synthesis in cultured cells. The influence of beta-aminopropio-nitrile. Proc. Soc. exp. Biol. Med. 130, 451-454.

BARNES, M. J. (1969). Ascorbic acid and the biosynthesis of collagen and elastin. Bibltca Nutr.Dieta 13, 86-98.

BATES, C. J. & LEVENE, C. I. (1969). The effect of ascorbic acid deficiency on the glycosamino-glycans and glycoproteins in connective tissue. Bibltca Nutr. Dieta 13, 131-143.

BATES, C. J., LEVENE, C. I. & KODICEK, E. (1969). The effect of scurvy on hexosamine-con-taining substances in healing wounds in guinea pigs. Biochem. J. 113, 783-790.

BERGMAN, 1. & LOXLEY, R. (1963). Two improved and simplified methods for the spectro-photometric determination of hydroxyproline. Analyt. Chem. 35, 1961-1965.

BURTON, K. (1956). A study of the conditions and mechanisms of the diphenylamine reactionfor the colourimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323.

GALAMBOS, J. T. (1967). The reaction of carbazole with carbohydrates. I. Effect of borate andsulphamate on the carbazole colour of sugars. Analyt. Biochem. 19, 119-132.

GOLDBERG, B., GREEN, H. & TODARO, G. J. (1963). Collagen formation in vitro by establishedmammalian cell lines. Expl Cell Res. 31, 444-447.

GREEN, H. & GOLDBERG, B. (1963). Kinetics of collagen synthesis by established mammaliancell lines. Nature, Lond. 200, 1097.

GREEN, H. & GOLDBERG, B. (1964). Collagen and cell protein synthesis by an establishedmammalian fibroblast line. Nature, Lond. 204, 347-349.

GREEN, H. & TODARO, G. J. (1967). The mammalian cell as differentiated microorganism. A.Rev. Microbiol. 21, 573-600.

GRILLO, H. C. & GROSS, J. (1967). Collagenolytic activity during mammalian wound repair.Devi Biol. 15, 300-317.

JUVA, K. & PROCKOP, D. J. (1966). Modified procedure for the assay of H3 or C14-labelledhydroxyproline. Analyt. Biochem. 15, 77-83.

KOCHHAR, D. M., DINGLE, J. T. & LUCY, J. A. (1968). The effects of vitamin A (retinol) oncell growth and incorporation of labelled glucosamine and proline by mouse fibroblasts inculture. Expl Cell Res. 52, 591-601.

MACEK, M., HURYCH, J. & CHVAPIL, M. (1967). The collagen protein formation in tissue cul-tures of human diploid strains. Cytologia 32, 426-443.

PRIEST, R. E. & BUBLITZ, C. (1967). The influence of ascorbic acid and tetrahydropteridine onthe synthesis of hydroxyproline by cultured cells. Lab. Invest. 17, 371-379.

PRIEST, R. E. & DAVIES, L. M. (1969). Cellular proliferation and synthesis of collagen. Lab.Invest. 21, 138-142.

PROCKOP, D. J. & KIVIRIKKO, K. I. (1968). Hydroxyproline and the metabolism of collagen. InTreatise on Collagen (ed. G. A. Ramachandran) 2A, pp. 215-246. London and New York:Academic Press.

PROCKOP, D. J. & UDENFRIEND, S. (i960). A specific method for the analysis of hydroxyprolinein tissue and urine. Analyt. Biochem. 1, 228-239.

SCHAFER, I. A., SlLVERMAN, L., SULLIVAN, J. C. & ROBERTSON, W. VAN B. (1967). Ascorbicacid deficiency in cultured human fibroblasts. J. Cell Biol. 34, 83-95.

SCHAFER, I. A., SULLIVAN, J. C , SVEJCAR, J., KOFOED, J. & ROBERTSON, W. VAN B. (1968).Study of the Hurler Syndrome using cell culture: Definition of the biochemical phenotypeand the effect of ascorbic acid on the mutant cell. J. din. Invest. 47, 321-328.

682 C. I. Levene and C. J. Bates'

SCHILLER, S., SLOVER, G. A. & DORFMAN, A. (1961). A method for the separation of acidmucopolysaccharides: its application to the isolation of heparin from the skin of rats. J. bio!.Chem. 236, 983-987.

STERN, B., GLIMCHER, M. J. & GOLDHABER, P. (1966). The effect of various oxygen tensionson the synthesis and degradation of bone collagen in tissue culture. Proc. Soc. exp. Biol. Med.121, 869-872.

TODARO, G. J. & GREEN, H. (1963). Quantitative studies of the growth of mouse embryo cellsin culture and their development into established lines. J. Cell Biol. 17, 299-313.

UDENFRIEND, S. (1966). Formation of hydroxyproline in collagen. Science, N.Y. 152, 1335—i34°-

WOESSNER, J. F. (1968). Biological mechanisms of collagen resorption. In Treatise on Collagen(ed. G. N. Ramachandran), pp. 253-330. London and New York: Academic Press.

(Received 18 March 1970)

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