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Philip Morris

Vitamin A, Carotenoids and Cell Function

Date: 19650000/P
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VITAMIN A, CAROTENOIDS AND CELL FUNCTION BY J. T. DINGLE* AND J. A. LUCY* Straugeways Research Laboratory, Cambridge (Received i February 1965) Membranes and excess of vitamin A (z) Introduction . (2) Structure and physical pro- perties of vitamin A . (3) The erythrocyte . . . (4) Mammalian fibroblasts. . (5) Cell organelles . (6) Tissues cultivated in vitro . (7) Fungi, bacteria and viruses . (8) Conclusions . . . III. Deficiency of vitamin A . . (s) General considerations (2) Mucopolysaccharide synthesis (3) Steroid synthesis . . (4) Biological oxidations . . (g) Organ cultures . . . CON'TENTS 422 IV. Membranes and electron transfer . 438 423 (z) Polyene molecules . . 438 423 (z) Vision . . . 441 (3) Olfaction and taste ~ 443 423 (4) Respiration compared aith 425 photosynthesis . . . 443 428 (5) Vitamin A and electron trans- 429 fer . . . . . 445 432 ' V. Active forms' of vitamin A. 434 449 43S VI. Condusions . 450 435 435 VII. Summary . 45= 435 436 VIII. References . 453 437 438 IX. Addendum . 458 I. INTRODUCTION This article is primarily concerned with a discussion of the interactions and possible functions of vitamin A within biological membranes. No attempt has been made to review all recent work on other aspects of the biochemistry of the vitamin, particularly since Olsen (1964) has lately reviewed investigations on the biosynthesis and meta- bolism of carotenoids and vitamin A. Trom the studies on the mode of action of excess of vitamin A that are discussed in Section II it has become increasingly apparent that vitamin A interacts strongly with the lipoprotein membranes of cells and intracellular particles. In general terms, compounds that are unable to prevent the symptoms of vitamin A deficiency are un- able to produce the effects characteristic of excess vitamin A, and we have proposed ' previously that membranes might also be a physiological site of action of the vitamin (Dingle & Lucy, t96z). It is thought that this suggested action may at least partially account for the many and varied symptoms of vitamin A deficiency, some of which are discussed in section III. Section IV includes a fairly detailed discussion on the electronic properties of polyenes, photosynthesis and respiration. It seems to us that these topics provide circumstantial evidence which suggests that vitamin A or a derivative may be con- cerned in sub-cellular electron transfer. Since the provision of energy at the right time and in the right place is essential for normal cellular functions, involvement of • Member of the External Staff of the 1ledical Research Council. i ; t
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Vitamin A and cell function 423 vitarrtin-A in mitochondrial reactions might explain many of the changes in morphology and metabolism observed in acute deficiency. Some readers may consider parts of section IV to be irrevelant to the problems of the mode of action of vitamin A. It is pertinent to refer, however, to the remarks made by Morton (iq6i) concerning an article of Green & Lester (iq3q) on mitochondria. Morton commented that 'It will be seen that the review by Green and Lester is an entrancing blend of fact and fiction, using the word 'fiction' to mean that which is imagined or invented. The field of mitochondrial structure in relation to electron transport lends itself to bold flights of imagination, of which perhaps it can be said the more the merrier, so long as they give rise, sooner rather than later, to crucial experiments.' The systemic mode of action of vitamin A has long remained a puzzl8 at the biochemical, level. It is hoped that the present article, though speculative in part, may suggest some new lines of fruitful investigation. , II. MEMBRANES AND EXCESS OF VITAMIN A (i) Introduction In this Section, relatively simple systems are considered before more complex ones. We shall discuss first some of the physical and chemical properties of vitamin A, then pass to its action on artificial membranes; the membranes of er}throc}-tes, fibroblasts and subcellular particles, and so to some of the actions of excess of the vitamin on organized tissues. By this arrangement, we have tried to present the reader with a rational interpretation of the salient features of the papers under discussion. (2) Structure and physical properties of v itamin A . The experiments by which the structure of the vitamin A skeleton was elucidated over 30 years ago have been reviewed by AIoore (1957). Vitamin A is a lower iso- prenologue of the monocyclic carotenoid pigments which contains twenty carbon atoms, and may have a-CH2OH, -CHO, -COOH, or an ester group at the end of the unsaturated aliphatic chain (Text-fig. i)*. The presence of an extra double bond in the /3-ionone ring of vitamin A2 (3-deh3=droretinol) was first demonstrated by Morton, Salah & Stubbs (1947). A number of homologues of vitamin A are now known, but since most of these are without biological activity they will not be dealt with in detail. They have been excellently reviewed both by Sebrell & Harris (1954) and by Moore (I957)• The chemical structure of vitamin A confers certain physical properties which are important in the mode of action of retinol and related compounds in biological systems. Of particular significance in the physiology of vision are the stereoisomcric properties of these molecules. Zechmeister (1962) has pointed out that the carotenoids f In this article vitamin At is referred to as retinol, retinene as retinal, vitamin A acid as retinoic acid, and vitamin A= as 3-dehydrorctinol. This terminology accords u ith the detinitive rules for the nomen- clature of vitamins published by the Commission on• the \omcncl.iturc of Biological Chcmistry, tg6o (7. amcr. Chein. Soc. 82, 5575-84). For convenience, we have used the term vitamin A to refer non- specifically to the group of compounds, including retinol, retinal, retinoic acid, and the esters of vitamin A, which have biological activity. I;iot. Rev, 40
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J. T. DINGLE AND' j. A. Lucy and related multi-conjugated systems are unique among substances of low-moleeular weight in having very many possible geometrical isomers. Furthermore, these mole- cules are `morphologically sensitive' in the sense that a single trans-cis shift may drastically alter their overall shape. This feature is not shared by other types of ctim- pounds that are rich in steric forms: thus, the shape of a sugar molecule is but little modified bc- epimerization. The chemistry of the cis isomers of vitamin A has been re«e.ved by _lforton & Pitt (IgS7). The importance of cis-trans isomerism in vision has been demonstrated in the brilliant studies of Wald and his collaborators; for reviews see Wald (Ig6o) and Pitt & Morton (Ig6z,). In contrast, the role of the cis isomers in growth is less certain. The g-cis, II-cis, 9-, I3-di-cis acetates, and the II-, 13-di-cis acetate, have less than a quarter of the activity of the all-trans forms, though II-cis retinal has nearly 5o% of the biopotency of all-traiu retinyl acetate. The most active ci.t isomers are the I3-cis forms, the aldehyde and the acetate ester having go% and 75% respectively of the activity of the all-trans ester (Ames, Swanson & Harris, I955)• This last observation may be related to the ease of conversion of the terminal 13-cis double bond to the CHCH, C ~Hs IHs CH: , C•CH=CH-C=CH---CH=CH-C=CH-R CH= , C•CHs CH1 Text-fig. t. General chemical structure of vitamin A derivatives. R is CHsOH in retinol, CHO in retinal, COOH in retinoic acid; in retinyl esters, R is an ester group. trans configuration. Zechmeister (Ig6o) has suggested that the most important criterion for biological activity is whether or not an isomer has the right shape to fit on to an enzyme surface. To support this view, he quotes the fact that all-trans-y carotene and pro-y-carotene have equal biological activity in the rat, and that in the chick the pro-;•-carotene is in fact 25% more effective than the all-trans isomer (Greenberg et al., I949)• Other phy_ical properties of the vitamin A molecule which may be important in determining its biological function are its solubility in organic solvents, its thermal stability in an inert atmosphere and its instability in the presence of oxygen or air. The addition of small amounts of anti-oxidants such as hydroquinone or a-tocopherol reduces the lability to oxidation (Milas, 1954). Another important feature of the vitamin A molecule is the presence of a relatively bulky, lipophilic ring which is located at the opposite end of the rigid chain from the hydrophilic group. This struc- tural arrar•.ge:nent is probably responsible for certain vitamin A derivatives behaving in a similar manner to lipids such as cholesterol, which have a hydrophilic hydroxyl group at or.e end of the molecule. Both cholesterol and retinol orientate themselves at an air-water interface to produce a monolayer. Determinations have been made of the collapse pressure and the minimum area per molecule for retinol by Weitzel, Fretzdorff & Heller (rg32), who obtained values of 2g~4 dynes/cm. and 26A?. Bangham, Dir.gIe & Lucy (1964) obtained values of 22-5 dynes/cm. and 25A.Z for ' N ai 0 ~ O on A ~ ~ IS
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t~e mole- ~ ahift may ipo of com- ~ is but little a A b.s been tated ;n the 0) $ntt Pitt & ~Cfwn. The t k" than a Orly So% of Wt the i3-fu .tively of the . Observatton bond to the , resinot. 4U t important shape to fit t all-trans-y i that in the rans isomer nportant in its thermal ygen or air. -tocopherol :ture of the ig which is This struc- ,:s behaving ic hydroxyl themselves cn made of )y Weitzel, and 26A.Z. 1 25A.z for ITitamin A and cell function 425 retinol and also found similar values to those of Weitzcl et alL for other compounds in the vitamin A series. Certain derivatives, such as anhydro-vitamin A, were unable to form films but behaved as non-spreading oils. I Bangham et al. (1964) aalso investigated the penetration of lipid monolayers by the vitamin. They used a film composed of equimolar quantities of lecithin and cholesterol, as a simple model for the lipid components of the plasma membrane of erythrocytes. Penetration of this monolayer at 12 dynes/cm. above the collapse pressure of retinol was studied, and retinol, retinoic acid and 3-dehydroretinol were found to cause the greatest increases in surface pressure on penetration of the lipid film. Retinol was the only compound of those investigated that was able to cause both a large increase in surface pressure at constant area and also a large increase in area of the lecithin- cholesterol monolayer at a constant surface pressure of 3o dynes/cm. Esterification of retinol greatly reduced or completely abolished its surface-active properties; the anhydro form of the vitamin and the methyl ether were inactive. These experiments therefore demonstrated that retinol is a powerful surface-active agent. It is interesting that decreasing the length of the side-chain of vitamin A resulted in greatly decreased ability to penetrate a lipid monolayer held at constant pressure. Hydrogenation of the unsaturated bonds, which produces a flexible side-chain also decreased surface activity. Replacement of the terminal hydrosy l group by a carboxyl groiup, which may be thought to increase the hydrophilic properties of the molecule, did not alter penetration of the lecithin-cholesterol film at a constant area though the ability to increase the area of the film at constant pressure was diminished. Inclusion of an extra double bond in the ring system as in 3-dehydroretinol reduced the expan- sion in area on penetration of a lecithin-cholesterol monolayer at constant pressure. Thus, the molecular specificity for surface activity observed in this model system was similar to that required for reversing hypovitaminosis A in animals. The ester and ether derivatives, which will be discussed later, and retinal, constitute exceptions to this generalization. Since these experiments were carried out in daylight, it is possible that partial conversion of the all-trans aldehyde to cis-isomers occurred. The penetra- tion and stability of isomers of retinal and retinol are now being investigated in this laboratory. (3) The erytlirocyte Although vitamin A is essential for nArmal growth and function, an excess of the vitamin produces many pathological changes. For example, the isolated erythrocyte is lysed by added retinol (Dingle & Lucy, i962); this has been confirmed by Kinsky (1963). We found that cells were rapidly destroyed when treated at 3?° C. with io fig./ml. of retinol. The haemolytic action of the vitamin was observed with eyrthro- cytes from rabbit, pig, ox, rat and man. Rapid l3•sis, which was inhibited by serum proteins, did not occur in the cold although potassium was lost and some lysis «-as observed after i hr. The molecular specificity for the action of the vitamin on crythrocytes has becn investigated (Dingle &- Lucy, ig62). Only weak activity was possessed by hydrogen- ated retinol, oxidized retinol, the anhydro derivative of retinol, 3-dehydrorctinol and . -' _
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3-dehydroretinal. fl-Ionone, geraniol, citral,:;plittol. and dodecanol were without activity at the concentrations tested. Both retinol and retinal were io times more effective in producing lysis than the related C14-: and _C27-alcohols and aldehydes that have shorter and longer side-chains respecti~-eh Tl~ie criteria for activity were the presence of the lipophilic ring system, containing-only one double bond, connected to a polar end-group by a rigid side-chain consistingof four conjugated double bonds. In general, haemolytic activity is possessed onh-. hy.those molecules that have biologi- cal ac ~'v~' y and are able to prevent the symptoms of;y_itamin A deficiency. The palmi- tate and acetate esters of the vitamin, which ar€-non-haemolytic, constitute a notable exception. NVe have suggested therefore that the:~cacy of the esters that is normally observed in nutritional experiments may depend;:on esterase activity; absence of lytic activity might reasonably be expected «•it~'.the palmitate ester since this is the normal storage form of the vitamin. The initial action of retinol on erythrocytes is believed to be a penetration and expansion of the cell membrane. Evidence for this view comes from the observations of Glauert, Daniel, Lucy & Dingle (1963), who investigated the sequence of changes in the fine structure of the cells during lysis by the.:vitamin, both by phase-contrast microscopy of intact cells and by electron microscopy of thin sections. They found that the initial effect of retinol, which occurred within r min., was the production of cells of bizarre appearance which had a greatly increased surface area as compared with untreated cells. Large indentations appeared on the surfaces of the cells and vacuoles were formed from the indentations'by a process which resembled micropinocytosis (Pl. i b). The cells then became spherical and, as breaks appeared in the membranes in (some of the cells, loss of haemoglobin began. Finally ghosts were produced that were no longer spherical but still contained numerous vacuoles (P1. i e). The appearance of breaks in the cell membrane and the consequent loss of haemo- globin. is a comparatively rapid process that onlh&rfiwsAte.mperatures above 20° C. At lower temperatures no breaks were seen, although the indentations and other morphological changes in the cell membranes tiveree observed. This is believed to be evidence of a two-step process in the haemolysis of the erythrocyte by retinol. The initial step is apparently a penetration of the membrane which occurs both in the cold and at physiological temperatures, although more rapidly at the latter. The large breaks observed above about 20° C. may be associated with oxidation of the polyene chain of retinol; this may result in co-oxidation of other elements of the lipoprotein membrane. The rapid haemolysis by retinol at 37° C. is inhibited by an equimolar quantity of a-tocopheryl acetate (vitamin E) (Lucy & Dingle, 1964). Electron micrographs, made in collaboration with Miss A. M. Glauert, of erythrocytes treated with retinol and vitamin E simultaneously showed an initial sequence of changes in shape similar to that seen with retinol alone (Pl. r d). After 15 min., however, the cells were still intact, though they contained vacuoles which may have been formed as a result of the initial expansion of the cell surface (P1. i e). It was concluded that retinol penetrated the erythrocyte membrane, both in the presence and absence of the a-tocophcryl acetate, but that this compound prevented the subsequent rapid rupturing of the cell membrane that occurs with excess of retinol alone.=
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Without - Cones more ,yda that were the connected 1e bonds. ~~te biologi- e palmi- a notable ,normally absence of this is the ,uon and Observations o#' changes ntrast They found uction of pared with and vacuoles Tnocytosis branes in that were of haemo- JbOVe 20° C. and other ieved to be ifetinol. The in the cold large breaks chain of membrane. quantity of Ephs,, made retinol and pC similar to lls were still a result of the I penetrated a-tocopheryl g of the cell Vitamin A and cell function 427 The stabilizing action of vitamin E may be related to its action in reducing the instability of a monolayer of lecithin and cholestcrol containing a high proportion of retinol (Bangham, Dingle & Lucy, unpublished observations). x-Tocopheryl acetate did not prevent penetration of retinol into the lipid monolayer but its presence in- hibited the subsequent slow fall in surface pressure attributed to oxidation of the retinol within the monolayer. Monolayers penetrated by retinol at 13 ' C. resembled those penetrated in the presence ofa-tocopheryl acetate at room temperature and were stable for at least i s min. As we have seen, erythrocytes treated with retinol at 8° C. are also stable for a considerable time; penetration of the cell membrane apparently occurs in the cold without causing haemolysis. Thus it seems that there are similarities between the inhibition of lysis by vitamin E and the inhibition associated with keeping the cells cold. The molecular specificity for inhibition of vitamin-A-induced lysis has been in- vestigated and it was found that although DL-a-tocopherol arid DL-2-tocopheryl acetate were very effective, the D-a-tocophery•I acid succinate was relatively ineffective and the phosphate ester was itself haemolytic (Lucy & Dingle, iq6.}). Hydroquinone was without effect in this system, even when quantities up to 5oo /ig./ml. were added simultaneously with 15 fcg./ml- of retinol. It -%vas thought that the failure of hydro- quinone to inhibit lysis due to retinol indicated that inhibition by the tocopherols tnay not be the result of neutralization of free radicals and the breaking of auto- oxidative chain reactions. This conclusion was supported by the fact that the acetate ester of a-tocopherol was particularly effective in inhibiting lysis, since the ester does not contain a hydroxyl group that can yield hydrogen and thereby neutralize free radicals. Compounds in the vitamin K series were also tested and vitamin Kl was found to inhibit retinol-induced lysis effectively, while menadione (vitamin Ii3), which has the same quinone structure as vitamin Kl but no isoprenoid side-chain, was inactive. 2-Methyl-4-amino-i-napthol (vitamin Kj was also inactive. Ubi- quinone-3o, which has a lQng side-chain, was a very effective inhibitor of rapid lysis despite the fact that the anti-oxidant activity of ubiquinone-3o is much less than that of a-tocopherol (Green et a1L iq6i). Two other long-chain compounds, squalene and phytol, which have no activities as vitamins, were capable of inhibiting the lysis by retinol. We concluded from these experiments on the relationship of molecular structure to inhibition of lysis that the inhibitory activity may be associated with the properties of the long isoprenoid chains of the active compounds. Erythrocyte membranes that contain exogenous retinol but are prevented from lysing rapidly by the presence of a-tocopheryl acetate or by low temperature have demonstrably altered properties. Thus, the addition of o•5°;, serum albumin to such cells caused virtually immediate and complete lysis, apparently resulting from com- bination of the added protcinwith moleculcs of rctinol alread.• pres ent in the membrane. These experiments led us to suggest that one of the physiolo-ical roles of vitamin A might be to stabilize membranes by acting as a cross-linking a;c:nt between the lipid ~and protein, since the vitamin can be strongly bound by both molecular species (Lucy S: Dingle, 1962; Lucy & Dingle, 1964). In contrast to the observations on the haemolytic action of retinol, treatment of ,
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428 J. T. DINGLE ,--%-D J. A. LzcY erythrocytes with low concentrations of retinol (o•I-I•o frg..'ml.) increased theresist- ance of rabbit, rat, pig, chicken and human er.-throcytes to hypotonic haemolysis. Protection was given by retinoic acid, vitamin A methvI ether and retinal, while the palmitate ester, 3 apo-/3-carotenal,,8-ionviidene acetaldehvde,,8-ionone and citral had little or no effect on hypotonic haemolysis. The protective action of the vitamin was prolonged either by keeping the cells cold or by the presence of x-tocopheryl acetate. The mechanism of this action of vitamin A is not clear, but one possibility suggested was that stabilization towards hypotonic lysis mav be associated with expansion of the surface of the erythrocyte by the vitamin, causing a change in the elasticity of the membrane (Lucy & Dingle, 1964). These studies on lipid monolayers and erythrocytes strongly indicate that retinol, unlike many closely related compounds, can interact strongly with natural and arti- ficial lipid membranes. It has been suggested that molecules which, like retinol, may control cellular functions by interactions with lipoprotein membranes might be suitably described as 'membrane-active' (Lucy, z964b). The initial penetration and expansion of the membrane by retinol are followed by various secondary effects that depend on the structure and stability of the vitamin-lipid complex and on the presence of suitable oxidizing agents. In the follon-ing Sections, the actions of vitamin A on the fine structure of fibroblasts, mitochondria, lysosomes, bacteria and viruses are discussed ; it is thought that the primary action in each of these systems is penetra- tion of the lipo-protein membranes. (.}) Mammalian fibroblasts The addition of retinol to fibroblasts suspended in buffered saline at 37° C. caused local distensions in cell membranes which finally disintegrated (D'ingle, Glauert, Daniel & Lucy, 1962). The nuclear membrane also showed signs of damage and the cytoplasm contained many unusually large granules; the mitochondria of the sus- pended cells became swollen and their internal structure distorted. These observations demonstrated that excess of the vitamin disorganizes the structure of many different membranes. In these experiments the cells took up the vitamin very rapidly, and the drastic nature of the changes produced often obscured finer details and made it impossible to follow progressive changes in the structure of the membranes. 'Recently, the action of the vitamin on the fine structure of fibroblasts grown in tissue culture as monolayers has been investigated (Daniel, Dingle, Glauert & Lucy, unpublished). Fibroblasts growing in a medium containing protein react relatively slowly to retinol since the free vitamin is not immediately available to the cells. Un- treated cells in control cultures had the typical fine structure of mammalian fibroblasts that are active in collagen synthesis (Pl. 2a, b). Fine fibrous material typical of fibro- blasts was visible just beneath the plasma membrane and parallel to it. After treatment with retinol there was little change in the appeatance of the nuclei of the cells during the first 3 hr. growth, and cells undergoing typical mitosis were observed. Approxi- mately 9 hr. after treatment, the cisternae of the endoplasmic reticulum began to disintegrate and were no longer visible in many cells. This disappearance of granular endoplasmic reticulum was accompanied by a marked increase in the number of free !
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'thi•resist- tmolysis. while the citral had jtgmin was i acetate. juggested nsioa of ity of the ,t retinol, and arti- ino1, may might be #ion and effects on the vitamin viruses penetra- induced by insulin. Many myelinated bodies composed of concentric arrays of membranes were present in the fibroblasts 'cultured with retinol. This m'ight reflect an incorporation of the vitamin into the membrane components of the growing fibroblasts and a resulting increase in the membrane area. On the other hand, the structures may not be pro- duced as a direct result of the action of the vitamin, since similar bodies have been observed, for example, by Karrer (1966) and Vickerman (1962) in degenerating cells and in cells that have ingested bacteria or other substances. Little change was found in the oxygen uptake of the fibroblasts for the first 6 hr. of Q caused s exposure to retinol, but between 6 and 9 hr. there was a rapid decline in respiratory made it .1 skin grown in culture in the presence of vitamin A, but not in the dermal cells. The and the ~ chondria were also noted by Fitton Jackson & Fell (1963) in the epidermis of chicken different of Iate-foetal mouse bones treated with retinol in vitro (Fell, i964a). Swollen mito- tions ~ Lucy & Fell, i96i); Glauert & Fell observed swollen mitochondria in the osteoblasts the sus- was observed in chick limb-bone rudiments treated in culture with retinol (Dingle, nd the ; swelling which became apparent after 6 hr. (cf. Pl. zc). A depression in respiration lauert, ~ -activity. This depression in respiration was probably correlated with the mitochondrial respiration of these skin explants was not studied. anular mal'an cclls hav bcen confirmcd b st 1' f th 1' t t' f 1 u o e c irec ac ~ e i t t n to I Most of tlic diverse actions of vitamin A on the membrane systems of living mam- pproYi- z i` -: (S) Cell organelles a~ during ~ ttment I however,-no_direct evidence to support this_suggestion_ ffbro- ; cytolysomes may represent a less stable or perhaps 'leaky' form of lysosome. There is, oblasts ` lysosomes both in viro and in vitro (Dingle, 1963), it is tempting to speculate that the Un- i from vitamin A-treated tissues (Fell & Dingle, 1963) and altering the stability of atively ~ increased considerably. In view of the action of retinol in releasing lysosomal enzymcs wn in ~, Although dense 'lysosomal' granules were visible throughout the time that the Lucy, fibroblasts were cultured with retinol, the number of cytolysomes (\ovikoff, 1963) of free isolated organclles; these investigations will now be reviewed. breaks were visible in the membranes in the sections. However, small specialized invaginatioris of the plasma membrane were more frequently observed in retinol- treated fibroblasts than in the controls. These small invaginations, and those seen in studies of erythrocytes (Glauert et-al- 1963), are reminiscent of the observations of '~:..- 429 ` 5- Vitamin A and cetl unction ribosomes, ibme of which were in organized and apparently helical aggregates (see P1. zd). These observations may indicate that a release of ribosomes from the membranes'pf endoplasmic reticulum occurs under the influence of excess retinol; Fitton Jackson & Fell (1963) found that few profiles of the endoplasmic reticulum remained iri -the basal cells of chick epidermis after 6-7 days' cultivation with excess of vitamin -A. The Golgi vesicles of the fibroblasts appeared sK•ollen and empty after growtti for only i hr. in the presence of the vitamin. There also seemed to be an increase in the number of micro-tubules. The plasma membrane remained intact throughout the period of culture and no • Paul & Pearson (i96o), and of Barrnett & Ball (i96o), on the ultrastructural changes ~ ~ . . .. . - . . - w y e es ion o 1c I i amm on u 0
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430 J. T. DINGLE AND j. A. Lucy (a) lllitochondria The addition of retinol in concentrations between 2 and io,rig./mli to mitochondria isolated from rat liver caused rapid swelling at 37° C. (Lucy, Luscombe & Dingle, 1963). This concentration range of retinol is very similar to that for thyroxine-induced swelling of mitochondria (Lehninger, Ray & Schneider, i959). The effect of the vitamin on isolated mitochondria was sharply temperature-dependent. The action of retinol was also tested on mitochondria isolated from other tissues. Kidney mitochondria showed approximately So°/a and heart mitochondria approximately 25 °,/0 of the sensi- tivity of those from liver, while spleen and brain mitochondria were relatively un- affected. Comparison of the relative effectiveness of retinol with that for thyroxine (Tapley & Cooper, 1956), showed similarities in tissue specificity. The actipn of retinol in causing rapid swelling of liver mitochondria was confirmed by Wang, Slater & Dartnall (1963). They found that retinol had no effect on mitochondria isolated from retina, however, and they also observed that the organelles obtained from retina were less sensitive than liver mitochondria to the action of added thyroxine and calcium. The molecular specificity of the vitamin's effect on mitochondrial swelling has been studied (Lucy et al. 1963). The only compound with a comparable activity to that of retinol was 3-dehydroretinol; the palmitate and acetate esters, the methyl ether, and the anhydro derivative of retinol, were all inactive. Retinal and retinoic acid were only weakly active. Increasing the length of the side-chain of the molecule greatly interfered with the ability of derivatives of vitamin A to cause mitochondrial swelling. The hydrogenated derivative was slightly active; geraniol, citral and ,8-ionone were inactive. Derivatives inactive at 37° C. were also inactive in the cold. Cyanide at a concentration of z-3 mivt inhibits the swelling action of a variety of agents including thyroxine (Lehninger & Ray, 1957; Chappell & Greville, 1958), but it did not prevent the swelling caused by retinol. This inactivity of cyanide sug- gests that the action of the vitamin, unlike thyroxine and similar swelling agents, is not dependent on respiration. The swelling induced by retinol more closely resembled that caused by Fe' ions than by thyroxine, since i mM sodium cyanide produced only partial inhibition (io-So%) of the swelling induced by Fe++ ions (Hunter et al. 1963). These workers demonstrated that there'was a close correlation between lipid peroxide formation ,in swelling and lysis induced by Fe' ions. Thus anti-oxidants, including a-tocopherol, almost completely inhibited lipid peroxidation and swelling caused by 5o f.,Nt Fel ion; we have shown that mitochondrial swelling caused by retinol is inhibited by a-tocopheryl acetate (Lucy & Dingle, 1964). The swelling of mitochondria induced by both thyroxine and retinol are inhibited by serum albumin; a concentration of q4 mg./mI- of albumin inhibited swelling caused by iopg./ml. retinol by approximately 8o%, during incubation for 5 min. at 37° C. Wojtczak & Lehninger (i96i) demonstrated that the inhibitory action of albumin on thyroxine-induced swelling was due to the trapping by the protein of an iso-octane- soluble material, designated U factor. This factor, which appears to be a mixture of saturated and unsaturated fatty acids, is formed in mitochondria under the influence i
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pndria Dingle, induced vitamin ~( retinol ondria sensi- Un- vroxine ion of ,a& Slater 6ted from were cium. L" been to that of * ether, g"c acid tU molecule ondrial Cjtntl and in the _rr~aticty of 4 , i95$) iude sug- is not 1~ teembled ~_~~roduced ffunter et al. et;;n lipid ttt#-oxidants, uad swelling C caused by :re inhibited lling caused :tt.'st 370 C. albumin on _.ctane- s hiixture of !c tnftuence t , r Vitamin A and cell function e 431 of thyroxine. Serum albumin is able to trap both U factor and retinol, and thus prevents the mitochondrial swelling produced by these two agents. (b) Zysosomes de Duve and his collaborators (for a review, see de Duve, r959) showed that liver contains a number of acid hydrolases associated with particles that sediment between mitochondria and microsomes. These granules, which are bounded by a lipoprotein- membrane, were termed lysosomes. Particles similar to liver lysosomes have now been isolated from almost all tissues and cells, the only apparent exceptions being mature sperm and erythrocytes. The normal functions of these organelles are as yet only partially understood, and vitamin A has proved a very useful tool in investigating the factors that control the release of the lysosomal enzymes. Retinol was the first compound of physio- logical importance to be shown to have an action,both on the release of lysosomal enzymes in the living cell and also on the isolated organelles (Dingle, i96i; Fell & Dingle, 1963). The initial observation of the action of retinol on the release of lysosomally bound acid protease was made on a`mitochondrial' fraction isolated from rat liver. It has since been found that the sensitivity of lysosomal particles in vitro to retinol is reduced if the preparation is contaminated with other organelles. In partially purified preparations, the vitamin is active in quite low concentrations These observations have been extended to lysosomes isolated from rat brain, kidney and spleen and to the release of ribonuclease, aryl sulphatase and acid phosphatase (Dingle, unpublished tivork). Retinol released enzymes from all these preparations, although not to the same extent in all tissues. Furthermore, the enzymes were not released in the same relative proportions from lysosomes of different origin. Ribo- nuclease was the enzyme most readily liberated from the organelles of rat kidney and brain; aryl sulphatase was less readily released than the other enzymes from the lysosomes of rat liver. Lysosomes of different tissues also vary in morphology, in stability to various stresses, in their response to lipid soluble compounds, and in the activity of their hydrolases. The term 'lysosome' appears to cover a number of particles with similar properties that differ functionally from one tissue to another, or even at different times in the same tissue. -The specificity of the action of vitamin A on lysosomal preparations was investigated by Fell, Dingle & Webb (1962), who examined a number of compounds, including derivatives of vitamin A, terpenes and alcohols, for the ability to release an acid pro- tease from lysosomes isolated from rat liver, and to reproduce the effects of vitamin A ~ on embryonic chick cartilage in culture (see bclow). They found that two-thirds of the bound proteasen-as released from the lysosomes by o-7ttmole of retinol/ml. Of thc other compounds studied at this concentration, only rctinoic acid had an effect comparable to that of rctinol; most of the other derivatives were inactive while the tcrpcncs had approximately 25 % of the activity. In general the specificity was similar to that found for erythrocytes and mitochondria (see above). fn organ culture at a'conccntra- tion of o•oi jimole(m1., none of the compounds examined except rctinoi and retinoic ' 25010$9477
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acid had. a significant effect on the metachromatic staining properties of the explants; the effects of these two compounds were identical. The precise mode of action of vitamin A on lysosomes is still not clear, but it seems probable that, like its action on lipid monolayers, erythrocytes and mitochondria, the primary action of retinol is to penetrate the lysosomal membrane. However,'the in- crease in surface area and formation of vacuoles that occur in erythrocytes have not been seen in lysosomes. Further, no marked swelling has been demonstrated similar to that seen in mitochondria. Electron micrographs of isolated lysosomes treated with retinol show some decrease in electron density that presumably reflects a loss of material. The action of retinol on erythrocytes and mitochondria also differs from that on lysosomes, since no inhibitory action of a-tocopheryl acetate has been observed in the lysosomal system, which suggests that oxidation of the vitamin may not be con- cerned in the release of the bound enzymes. Retinol has been recovered from lysosomes treated in vitro with the vitamin, and also from lysosomes of animals suffering from hypervitaminosis A (Dingle, Moore & Sharman, ig63). In the latter experiments, however, the vitamin was not preferen- tially concentrated in the lysosomal-rich fraction. A. C. Allison (personal communica- tion), using fluorescent microscopy, has recently demonstrated the presence of the vitamin in the lysosomes of cells treated in culture with vitamin A. Lysosomes have been isolated from hSpervitaminotic animals and found to be less stable during incubation at 37° C. than those from control animals of the same age and sex (Dingle et al. 1963). By the use of standardized homogenization and sedi- mentation techniques, it was also found that the ratio of 'free' to 'bound' enzyme activity within liver tissue was much higher in vitamin A-treated animals (this ratio is a measure of the stability of lyosomes to mechanical stress). These observations have been confirmed by Weissman & Thomas (1963) in experiments with rabbits and guinea-pigs. The stability of the lysosomes of rat kidney was not, however, greatly affected by hypervitaminosis A (Dingle et al. 1963). A different tissue specificity has been seen in vitamin E deficiency; the stability of lysosomes in the kidneys of deficient rats was much decreased under the conditions of the experiments but no such effect was observed in rat liver lysosomes (Dingle, Moore & Sharman, unpublished work). The changes in lysosomal stability in the kidney were correlated with the very rapid post-mortem autolysis of this organ in vitamin E deficiency. It is relevant that Roels, Trout & Guha (1964.) have recently observed that the stabi- lity of liver lysosomes in vitamin A-deficient rats is greatly impaired. Oral administra- tion of retinyl acetate to the deficient animals reversed this phenomenon. (6) Tissues cultivated in vitro _ In this subsection, the effects of the vitamin on certain organized tissues are con- sidered in the light of the interactions of the vitamin with membranes and with intracellular particles. Fell & Mellanby (iq5a) showed that, when the cartilaginous limb-bone rudiments from 7-day chick embryos were grown in the presence of excess of vitamin A, meta-. chromatic material was progressively lost from the matrix, which rapidly disintegrated. 0 i I
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bot it seems ria, the ~ the in- have not imilar trated with tt loss of from that observed in t be con- d to be less the same age ion and sedi- d' enzyme Is (this ratio observations rabbits -and rer, greatly pecificity has of deficient no such effect ished work). e very rapid Vitamin A and cell function 433 Later it was found that. some of the effects of the vitamin on the extracellular material of cartilage could be simulated by the action of papain (Fell & Thomas, ig6o). It was then shown that vitamin A causes a loss of hetosamine from the extracellular material of the cartilaginous explants, as well as alterations in the R\A to Dti'A ratio of the cells (Dingle, Lucy & Fell, ig6i). Some of the changes seen in the presence of the vitamin can also be duplicated by hypo-osmotic treatment of the, normal limb-bone rudiments followed by incubation at an acidic pH (Lucy, Dingle & Fell, rg6r); this treatment releases an acid protease, which is now known to be present in the lysosomes, and most of the changes in the extracellular matrix can be ascribed to the action of this enzyme on the protein moiety of the protein-polysaccharide complex. Further experiments on the effect of vitamin A on cartilaginous rudiments in organ culture showed that not only is more of the h•sosornal protease released into the culture medium from vitamin A-treated rudiments, but that the treated explants often contain more off the enzyme than the larger control rudiments; a compensatory synthesis of lysosomal enzymes may therefore have occurred under these conditions (Fell & Dingle, 1963). From these results it was concluded that the vitamin probably acts on the lysosomes of the living chondrocytes in the same way as it acts on the isolated organelles, i.e. that it penetrates the 1}-sosomal membranes and so releases the acid protease that is responsible for the degradation of the matrix. Rapid loss of glycogen from the chondrocytes is another feature of the action of retinoll on tissues cultured in vitro, and it has been suggested (Dingle, 1963) that this may by related to the presence of an a-(i-q.)-glucosidase within lysosomes (Lejeune, Thines-Sempoux & Hers, 1963). The series of experiments which led to the hypothesis that vitamin A acts on the organelles of the living cell has recently been reviewed by Fell (ig64b) and hence will not be discussed in detail here. Support for this hypothesis has come from studies on the action of inhibitors on the vitamin A-induced changes. It is possible to inhibit the effect of the vitamin on cartilage matrix in two ways. First, some physiologically active compounds have been shown to stabilize lysosomal particles and diminish the release of the hydrolases. Secondly, the active enzymes can be inhibited after their ' release but before they have degraded their macromolecular substrates. As an example of the first type of inhibitor, cortisol has been shown to antagonize partially the action of vitamin A on both embryonic fowl skin (Fell, rg6a) and embry- onic cartilage and bone in culture (Fell & Thomas, zg6z). This inhibition is apparently associated with a direct action of the steroid on lysosomes. de Duve, NVattiaux & Wibo (tg6i) found that cortisol inhibited the thermal activation of isolated lysosomes and Weissman & Dingle (Ig6I) showed that lysosomes isolated from cortisol-treated rats were more stable to the effects of ultraviolet irradiation than were those isolated from control rats. Further evidence that cortisone preserves and protects lysosomes under various conditions has been obtained both in this laboratory (Sledge & Dingle, ig64; Weiss & Dingle, ig63), and by other workers («'eissman ~C: '1'homas, tg6.}). Free lysosomal protease is inhibited by s-amino caproic acid (A1i, 1964). '1'his finding has been confirmed in our laboratory, and it has also been observed that the release of chondroitin sulphate from chick limb-bone rudiments which have been
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434 J. T. DINGLE AND J. A. Lucy exposed to hypotonic conditions is inhibited (Dingle, unpubl.). Degradation of cartij lage matrix by rctinol in organ cultures of chick limb-bones is diminished.by e-amino caproic acid at a concentration of o•int, while preliminary experiments indicate that bone resorption in the limb rudiments of mice is similarly reduced (Fell & Dingle, unpubl.). As already mentioned, liberation of lysosomal enzymes, and the consequent de- gradation of the matrix, caused a substantial reduction in the wet and dry weights and in hexosamine content of vitamin A-treated rudiments (Dingle et al. 1961). It seemed possible that excess of the vitamin might also inhibit the actual synthesis of protein and polysaccharide in cartilage. This point has been investigated. Dingle, Fell & Lucy (unpubl.) found that hydroxyproline and hexosamine are liberated into the medium from both control and vitamin A-treated explants, although the quantities released were smaller in the control than in the treated cultures. The total synthesis of hydroxyproline is apparently unaffected by excess of vitamin A. (7) Fungi, bacteria and viruses Kinsky (1963) has shon-n that whereas protoplasts of the fungus Neurospora crassa are rapidly lysed by polyene antibiotics, vitamin A (presumably retinol) did not significantly inhibit the growth of this organism, nor did it have an effect on the isolated protoplasts. However, protoplasts of Bacillus megaterium, strain KM, which were not affected by high concentrations of polyene antibiotic, were readily lysed by vitamin A. This observation has been confirmed by McQuillen, Glauert & Lucy (unpubl.) who independently found that a suspension of these bacterial protoplasts (io mg./ml.) treated with retinol (3o fcg./ml.) for 15 min. at 37° C were extensively lysed. Since both erythrocytes and the protoblasts of Neurospora, which are lysed by the polyene antibiotics, contain sterols, while sterols are absent from the insensitive protoplasts of Bacillus megaterium, Kinsky (1963) suggested that sterols must be present for the lytic action of polyene antibiotics. It appears that retinol differs from both these antibiotics and saponin (cf. Razin & Argaman, z963) in that it is able to lyse cells containing little or no cholesterol in their membranes. This is probably related to the observations of Bangham et al. (i964), who found that lecithin, not cholesterol, was primarily concerned in the penetration of lipid films by retinol. Blough (z963) studied the effects of various vitamin A preparations on the integrity and shape of certain viruses. The viral preparations from the treated eggs were very pleomorphic, with filamentous forms constituting approximately one-third of the specimens examined. Blough proposed that the changes in viral morphology probably reflected alterations in the physico-chemical structure of the cell membrane. He suggests that vitamin A may alter the packing in'the bimolecular lipid leaflet since `a linear orientation of the cholestcrol-phospholipid molecules would account for the production of viral filaments'. Alternatively, because of the high degree of unsatura- tion of vitamin•A, 'a sudden increase in Van der Waals forces between lipid molecules would cause a separating out of cholesterol from the leaflet'. The observations of Blough support the hypothesis that vitamin A has a function in the control of --:. N r-n 0 ti 0 co ~ ~ ~ 0
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Uent de- t$ and ssMed protein Fell Ec 16to the I Wtities synthesis a aassa did not ~it on the -1W, which olysed by ut & Lucy toplasts qttensively by the (nsensitive must be dWers from ia able to t probably i€hin, not r#inol. the integrity were very ird of the probably brane. He 1eAet since unt for the of unsatura- id molecules tions of c ,control of Vitamin A and cell function 435 membrane permeability (Dingle & Lucy, i96z; Lucy, i964a) and structure (Fell & Dingle, 1963), but the precise physico-chemical interaction of the vitamin with the } membrane lipids remains an open question. . (8) Conclusions Since membranes play such a major role in the structure and functions of cells, it is clear that any modification in their properties will have important biological con- sequences (cf. *illmer, ig6z). More than 30 years ago, Peters postulated the presence of a tenuous nettvork within the cells, the 'cytoskeleton', which co-ordinates the actions of the cell's enzymes. More recently, he has pointed out that an agent which can change the cytoskeleton might be expected to modify several enzymic reactions simultaneously (Peters, 1956). It seems that many of the widespread effects of excess of vitamin A may likewise result indirectly from its actions on the lipoprotein mem- branes of cells. The possibility that membranes may also be concerned in some of the physiological actions of the vitamin is considered below. III. DEFICIENCY OF VITAMIN A (x) General considerations ?12oore (1957) has -remarked that the effects of deficiency are so numerous and diverse that it is difficult to group them together in a clear and unified picture. _'S~ever- theless, he has provided a valuable table of lesions sustained in vitamin A deficiency. General effects are cessation of growth, failure of appetite, decline in body weight and infections in membranes. Specific abnormalities occur in the eyes, respiratory system, intestinal tract, urinary system, liver, skin, nervous system, bones and repro- ductive tissues; congenital abnormalities also occur. It is this very widespread action that makes it so difficult to extrapolate from particular observations on one system to any comprehensive mechanism for the biochemical action of the vitamin. It is clearL- not possible to present an exhaustive review of all recent work on the biochemistry of deficiency, and the reader is referred to the articles of Moore (1962) and Gloor & Wiss (1964) for more comprehensive discussions of this topic. In the following paragraphs, we shall consider certain features of deficiency that may depend, in some way, on interactions of the vitamin with membranes. (z) Muco~ol),saccharide synthesis An extensive study has been made by Wolf and his colleagues of the role of vitamin A in mucopolysaccharide synthesis. Wolf R: Varandani (ig6o) investigated the in- corporation in vitro of FS)sulphate into mucopolysaccharides by homogenates of rat colon. They observed that incorporation in preparations from vitamin A-deficient rats was onlc about half the normal. Retinol, retinal and retinoic acid, when added to the deficient homogcnates, restored incorporation almost to normal; vitamins D and h.rere inactive, and vitamin E only partially active in this system. Wolf, Varandani * Johnson (ig6t) traced the site of this action of vitamin A to an cnz}•me fraction of colon mucosa, and also obtained evidence indicating that the block in mucopoh•- :~~.~~~:. . ~ . . .
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4 436 • J. T. DINGLE AND J. A. Lucy saccharide synthesis associated with deficiency of the vitamin was in the synthesis of 3'-phosphoadcnosine-5'-phosphosulphate or 'active' sulphate (Varandani, Wolf & Johnson, ig6o). In later experiments, Wolf, Bergan & Sundaresan (1963) fractionated homogenates of rat colon and found that the mucopolysaccharide-synthesizing` activity resided mainly in a fraction containing particles that sedimented in 20 min. at 2o,ooog. Particles obtained from vitamin A-deficient rats showed greatly reduced mucopolysaccharide synthesis. Although retinol restored the activity of homogenates from deficient animals, this compound was much less effective with the particle preparations. Instead, a metabolite obtained from the intestinal lipids of rats fed with labelled retinoic acid re-activated the particles. Wolf et al. (1963) suggested that these observations indicate that retinol is not active itself in mucopolysaccharide synthesis but that it is first changed into an `active form'. Further work by Yagishita, Sundaresan & Wolf (1964) on an `active form' of vitamin A is discussed in Section V below. The possible role of vitamin A in the activation of sulphate has recently become the subject of some controversy, but this cannot be discussed in detail here. Pasternak, Humphries & Pirie (1963) have been unable to confirm the observations of Wolf and his colleagues, whereas the work of Rao, Sastry & Ganguly (1963) and Rao & Ganguly (1964) is, in general, consistent with that of Wolf. In a study of the biosynthesis of the hexosamine moiety of mucopolysaccharides by colon homogenates, Moretti & Wolf (ig6i), have concluded that the vitamin is apparently required, not only for the acti- vation of sulphate, but also for other processes in the synthesis of mucopolysaccharides. NVolf (1962) has commented that, in view of observations on the mode of action of excess of vitamin A on cartilage tissue made in our laboratory (cf. Section II), it is tempting to speculate that retinol, or better, an `active form' derived from retinol or retinoic acid, releases an enzyme (or enzymes) necessary for the biosynthesis of mucopolysaccharide from the particle fraction sedimenting at 20,000 g. He remarked that this process would then parallel that for the release of proteolytic enzymes from lysosomes under the influence of excess of vitamin A. Wolf also suggested, as an alternative, that vitamin A or a derivative could regulate the release of mucopoly- saccharide itself from the particles by changing their permeability; it may be recalled that it has been suggested that vitamin A may control membrane permeability to large molecules (Dingle & Lucy, 1962). In a theoretical discussion, Brown, Button & Smith (1963) have proposed the general hypothesis that the permeability changes associated with hypervitaminosis A or with deficiency of vitamins D and E may be due to a change in sulphation of the polysaccharides that are necessary for membrane structure. (3) Steroid synthesis Incorporation of cholesterol-4 14C into corticosterone is markedly depressed.in homogenatcs of the adrenal gland from vitamin A-deficient rats as compared with preparations from normal animals (Van Dyke, Wolf & Johnson, ig6o). The addition of retinol or,retinoic acid to the adrenal homogenates from deficient animals restored the normal formation of corticosterone from cholesterol. Since Halkerston, Eichorn
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af '~fEt ted iZing mia• uced nates iCle fed that ide ishita, ion V e the ternak, olf and fznguly of the & Wolf e acti- arides. action of I1),itis ta retinol or thesis of remarked les from td,asan _ inucopoly- U recalled eabilitv to Button & ty changes and E may be tor membrane depressed in eompared with I. The addition ~timals restored krston, Eichorn Vitamin A and celt function' 437 • . & Hechter (1959) found that the enzymes producing progcsterone from cholestcrol are located in mitochondria, Wolf (1962) has suggested that this effect on steroid biosynthesis might also be related to the action of vitamin A on membranes. (4) Biological oxidations Wolf, Lane & Johnson (1957) found that the incorporation of acetate-i-I'C, lactate-i 14C and glycerol-i, g 14C into liver glycogen was drastically reduced in vitamin A-deficient rats. At the same time, they observed no appreciable effect of deficiency on the incorporation of acetate into carbon dioxide, liver protein, aspartic acid or alanine, indicating no disturbance in the tricarboxylic acid cycle or in the reactions connecting it to glycolysis. They interpreted these results to mean that vitamin A is involved in the reversal of glycolysis between the triose and the glucose stage. It was also suggested, however, that the interference in the reversal of glycolysis may be of an indirect nature, possibly due to an influence on cell perme- ability or on the formation of a hormone controlling glycolysis. Wolf, Wagle, Van Dyke & Johnson (1958) noted that oxidative phosphorylation in the homogenates of liver was not affected by deficiency in that there was no shortage of high-energy phosphate or phosphorylating ability. Redfearn (1956) found, however, that the endogenous respiration of liver homogenates from deficient animals was .significantly higher than that of the controls. Furthermore, a marked difference was found in the activities of succinate-cytochrome c reductase; the mean value of the activity of the A-deficient group was only 59 % of that of animals receiving vitamin A. Vignais (1957) observed a considerable increase in the activity of TPNH-cytochrome c reductase, and a decrease in transhydrogenase in the mitochondria of rats deficient in vitamin A. Recently DeLuca, Manatt, Madsen & Olson (1963) have found that vitamin A deficiency in rats raises. the rate of oxidation of pyruvate, citrate, a-ketoglutarate, succinate, glutamate and fumarate, but not /.3-hydroxybutyrate and caprylate, by homogenates of liver, though not by kidney or heart preparations. Administration of retinyl acetate to deficient rats reduced oxidation to normal within 48 hr. It might be suggested that all the respiratory changes associated with deficiency that are dis- cussed here may be an indirect effect of the increased level of coenzyme Q that is known to be associated with deficiency of vitamin A (for a review, see Morton, 1961). However, the rapid response to the vitamin by the rats studied by DeLuca et al. appears to indicate that these workers may be studying a direct action. In the light of their experiments, DeLuca et al. suggested that vitamin A acts on a system common to all the tricarboxylic acid cycle substrates, such as electron transport, oxidative phos- phorylation, or mitochondrial structure, and that the lack of effect with /3-hydioxy- butyrate or caprylate may well by related to the surface-active properties of these two compounds. Seward & Hove (1964) liave confirmed that deficiency causes an increased rate of oxidation of a-ketoglutarate and succinatc in honiogrnatcs of rat liver, but they also observed that the increased rate is only temporary and not main- tained over a period of i' hr. At this point, it is relevant to note that there is a drerra%cd oxidation of succinatc in liver homngenatcs from hypervitaminotic rats (1Zay ,1 Sadhu, .~ 2501-089483 .. -
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438 J. T. DINGLE AND J. A. Lucy Igjq). Inhibition of succinate dehydrogenase activity. was also recorded by Rky-. & ..,., Sadhu, who concluded that hypervitaminosis A inhibits liver respiration b7 affecting the dchydrogenase or a step immediately following it. From all these observations we may conclude that vitamin A has some role in the oxidative reactions of cells, but the actual mode of action is far from clear. (5) Organ cultures Squamous metaplasia of secretory epithelia is known to occur in many glands, in- cluding the mouse prostrate, as a result of deficiency of vitamin A. Lasnitzki (1962) has found that similar changes occur when the prostrate glands of young adult mice are cultured in a chemically defined medium that does not contain vitamin A. The squamous changes can be suppressed completely by the addition of retinol to the medium. These experiments demonstrate that certain symptoms of vitamin A de- ficiency can be reversed by retinol, not only in the whole animal, but also in isolated tissues. In this instance, therefore, it is apparently not necessary to postulate that the effects of the vitamin are mediated by any kind of hormone action; vitamin A seems to act'directly on the mouse prostate gland. Organ cultures of the prostate thus provide an ideal closed system for the study of vitamin A deficiency, comparable to the cul- tures of embryonic chick cartilage that have previously been used in work on excess of vitamin A (see Section II (2), above). Cultures of the prostate might well be employed in attempts to investigate the uptake and cellular localization of the vitamin by means of autoradiography at the light and electron microscope levels, and experi- ments of this kind may shed further light on possible connexions between vitamin A deficiency and the action of the vitamin on membranes. Aydelotte (1963 a, b) has made an interesting study of the inhibitory action of citral on epithelial differentation in vitro. Citral was found to produce changes resembling those of vitamin A deficiency in cultures of chick oesophagus and cornea, and it also reduced the effects of added vitamin A. Aydelotte has suggested that both deficiency of vitamin A and treatment with citral stimulate mitosis in the chick corneal epi- thelium, while high concentrations of vitamin A inhibit mitosis. She also suggests that citral may prevent vitamin A from entering cells, or compete for active sites on the cell membrane. AA further study of citral, which may be a competitive inhibitor of vitamin A, would thus be valuable. IV. MEMBRANES AND ELECTRON TRANSFER (z) Polyene molecules Some physico-chemical properties of compounds in the vitamin A series have been briefly discussed above in Section II (2). We shall now consider in more detail the properties of the conjugated chain of alternating single and double bonds that is a common feature of these compounds and of the carotenoids in general. An important feature of such a chain is that delocalization or mobility of the zr-electrons applies not merely to the individual double bonds but to the chain as a whole. This electron mobility in the carotenoids has attracted attention for some time in relation to photo- I
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Vitamin A and cell function 439 '-synthesis, and it seems possible that electron transfer, associated with their polyene chains, might perhaps be a common function of both carotenoids and vitamin A derivatives. Thus the electron mobility of these compounds may be utilized in specific molecular complexes, present within specialized subcellular organelles, as essential features of photosynthesis, vision and the systemic actions ;of vitamin A. In view of this possibility, a number of papers are discussed in the following paragraphs that are concerned with observations and suggestions made in relation to the electron mobility of vitamin A and carotenoids. ~ Platt (igsg) has developed an idea, suggested earlier by Strehler, that light energy may sometimes be transferred from a photo-excited chlorophyll molecule to a caro- tenoid, rather than in the reverse direction. Platt proposed that such a process might be feasible if the carotenoid were in the form of a complex having excited states below the lowest excited state of chlorophyll. An electron donor molecule, D, is imagined to approach one end of the polyene chain of a carotenoid, while an electron acceptor, A, approaches the other. The resulting system ~ D.=-=-........ -=--.A would be a`trimolecular charge-transfer complex, D. carotene A.'. It is argued that, as a result of resonance stabilization, the absorption peak of the carotene molecule would be shifted to much longer wavelengths and hence the excited state of the complex could be brought below that of chlorophyll. Platt suggested that, if its energy is adequate, the D'.Car.A- excited state could then go easily by a radiationless transition to a separate configuration, in which the D' and A- molecules (ions or radicals, or both) move away, and the electrons in the polarized carotene aiolecule shift back to their free-carotene ground state arrangement. Thus carotene would behave as a kind of 'electron mediator' as suggested by Strehler. In connexion with Platt's hypothesis, Pullman & Pullman (1963) have remarked that the values of the energy coefficients of the highest occupied and lowest empty molecular orbitals of the carotenoids, including vitamin A, indicate that these compounds should be both excellent electron donors and electron acceptors. This would therefore support the idea of carotenoids behaving as electron mediators. Calvin (1939) has suggested that chlorophyll absorbs light to bring it to its lowest singlet excited state, and that the excited state can move around among the chlorophyll molecules until a point is reached where ionization occurs, when charge separation can take place. He has proposed a schematic molecular structure for a chloroplast lamella in which carotenoid and phospholipid molecules are packed between chlorophyll molecules. From a study of electron-transfer systems in certain photo-synthetic bacteria, Nishimura & Chance (i963) recently concluded that it is unlikely that carotenoids are situated on the main path of electron transfer in the photosynthetic and respiratory systems of these organisms because other carriers of electrons function normally in the carotenoid-less mutants of Rhodupseudunuonas spJreroides and Rl:odosphirilkun rubrum. However, changes in the carotenoid absorption spectrum induced by light or oxygenation were thought to be associated with the flow of electrons between carotenoid molecules and the other elcctron carriers. 1'he change in the c3rotcnoid 28 , Bt.,l. xe.•, {o . .. ~ . • . - . , U1 O 10 4
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r 440 J. T. DINGLE AUND J. A. Lucy steady state caused by addition of antimycin A or heptylhydroxyquinoline-N-oxide . also favoured this possibility. Jahn (ig6a) has put forward a theory of electron conduction through membranes, and of active transport of ions, that is particularly relevant to the work discussed in Section II above. He has suggested that membrane potentials may be primarily oxidation-reduction potentials resulting from the presence of oxidation-reduction enzymes on the two sides of a membrane, and that the system may be activated' by hydrolysis of adenosine triphosphate (ATP), which would serve as a source of electrons. Of particular interest in this scheme is the proposal that the two sides of the membrane are connected electronically by long lipoid molecules, containing alternating single and double bonds, which could bridge the lipoid film of the membrane and thus 'form a perfect pathway for electrons produced by an oxidizing enzyme on one side of the membrane and received by a reducing enzyme on the other side'. Carotene, astacene, retinene, crocetin and vitamin A are suggested as possible candidates for the conjugated lipid. Since this idea was proposed, much evidence has accumulated to indicate that retinol interacts with the lipid moiety of biological membranes (see Section II above). It is clear, however, that should retinol or a metabolic derivative of similar length function in the way proposed by Jahn, two or more molecules arranged in some back-to-back fashion would be necessary to extend right through a biological membrane from one side to the other. Little (1964) considers that the theoretical criterion for the occurrence of super- conductivity can be met in certain organic polymers. He has discussed details of an example in which the molecule consists of two parts, a long chain called the 'spine', and a series of side-chains attached to the spine at regular intervals. It is assumed that the chain is a conjugated system of double and single bonds; each side-chain is part of a dye molecule in Khich a positive charge resonates between two nitrogen atoms. Little proposes that, by appropriate choice of the molecules constituting the side- chains, the virtual oscillation of charge in the side-chains can set up an interaction between the electrons moving in the spine; theoretically this interaction can be made sufficiently attractive for superconduction in the chain to result. It is concluded that superconductivity could and should occur in such structures even at room tempera- ture. This conclusion is very interesting in relation to the properties of the conjugated chains of the carotenoids and derivatives of vitamin A since Little makes the point that there are many other possible structures, similar to the one discussed, which involve a semiconducting chain for the spine and a dye-like molecule for the side-chain that would also be superconducting. Some recent observations of Chapman & Cherry (xg6¢) are relevant to this discus- sion. These workers heated a crystal of ~B-carotene in a vacuum for several hours at a2o° C.; when an electric field greater than 4000 V./em. was then applied to the 'crystal, current pulses were observed despite the fact that the voltage was steady. The pulses were of constant frequency, occurred in the dark (frequency ~,Y cyc./sec.), and showed no signs of decaying over a period of several hours. On exposure to visible light, both the current and the frequency of the pulses were increased. Whereas the crystal in a vacuum showed current pulses at temperatures greater than g0° C.; ,J
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branes, ed in prirnarily ,ieduction sed' by tjectrons. ;oembrane ins single ynd thus one side C`arotene, ates for mulated rtttes (see derivative molecules through a of super- &Wls of an `spine', Wwmed that is part atoms. the side- itnteraction ±ud be made uded that Iennpera- Cbnjugated point that hicltt involve in that this discus- hours at ?ied to the steady. ~ cyc./sec.), -"sure to Whereas : ~an qo° C. , Vitamin A and cell functiort 441 F the pulses occurred at lower temperatures in the presence of oxvgcn and were even observed at room temperature. Chapman & Cherry have drawn attention to the fact that the variation in frequency of the pulses of /3-carotene with illumination is similar to the recording of spike discharges observed with a single optic nerve fibre of Limulus, and they cautiously raise the question of whether a mechanism of the type observed could initiate the passage of impulses from some receptor site, such as the retina, to the optic nerve. (a) Vision Many years ago Wald (1943) suggested that the role of vitamin A in vision may be an evolutionary development of the function of carotenoids in photosynthesis, since photoreceptor systems throughout the range of living organisms depend very generally upon carotenoids in various forms. More recently, in a discussion of why the caro- tenoids fulfil this role, Wald (i96a) has suggested that it is because of their peculiar capacity for geometrical isomerization. The ii-cis isomer of retinal is specifically concerned iri the visual cycles of~ molluscs and arthropods, as well as vertebrates. Wald therefore considers that this isomer may have been selected by each of the phyla since it possesses the highest photosensitivity, in the sense that it is photo- isomerized to all-trans retinal with the highest quantum efficiency, and hence at the highest rate. Although the role of retinal in vision is highly specialized, its site of action is never- theless intimately concerned with membranes. Interrelationships between the func- tion of retinal and membrane structures in the eye are shown by the work of Dowling & Gibbons (r96i). They observed that, during vitamin A deficiency, the highly ordered structure of the outer segment gradually breaks down, and degeneration begins with a marked swelling of the transverse, double-membrane disks, which become pinched off to form large vesicles and tubules. The anatomical degeneration of the outer segments of the rods appears to parallel the loss of the visual protein, opsin, that occurs in deficiency. Since opsin is a major component of the outer seg- ments, Dowling & Gibbons suggest that its loss may well be directly responsible for the breakdown of structure. In these experiments the fact was utilized that rats maintained on retinoic acid, though gro«•ing normally and otherwise in good condition, become extremely night blind and eventually completely blind from a deficiency of retinal which is needed for the synthesis of visual pigments. Dowling & Wald (i96oa) have commented on the close relationship between vitamin 3 and the elements of the eye in the following terms: 'for want of vitamin A the chromophore was lost; for want of its chromophore, the opsin was lost; for want of the opsin, the outer segment, was lost; for want of the outer segment, the rod was lost; for want of the rods, vision was lost; for want of its vision, the rat should soon be lost-and all for the want of vitamin A'. Hyono, Kuriyama, Tsuji & hIosoya (1962) have investigated the properties of a monolayer of rhodopsin at an air-water interface. When the film was kept at constant area and relatively low surface pressure (below 5 d}'nes,'cm.), exposure to a flashlight augmented a tendency for the pressure to increase spontaneously. After bleaching
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t 442 J. T. DINGLE AND J. A. Lucy of the rhodopsin, this augmentation of surface pressure following illumination no lorrger occurred.' It may perhaps be concluded from the studies of these workers that the ob- served spontaneous increase in surface pressure was associated with a low conversion of i i-cis retinal to the all-tranu form: a process that is rapid in the light. The significance of this and other observations given in the paper is, however, by no means clear. Rosenberg (1959) has discussed some theoretical and experimental aspects of photo- conductivity in j3-carotene. He prepared a glass from all-trans,8-carotene, which was photoconductive and which contained a mixture of stereoisomers. The photoconduc- tion action spectra of this glass indicated that absorption in the cis peak region (35ooA) was far more effective in liberating charge carriers than excitation in the first absorption band. In the course of these experiments, some anomalous observations were made which appeared to indicate that the carotene glass could behave as a source of current; it is possible that these observations may be related to those of Chapman & Cherry (1964) referred to above. Rosenberg, Orlando & Orlando (1961) have noted photo- conduction and semi-conduction in dried receptors of sheep eyes, but Brown, Gibbons & Wald (i963) consider that this photoconduction was far too slow to account for i.-isual excitation. Brown et al. suggested that a solid state phenomenon such as electron migration, if it exists in rods, would be expected to be limited to the double- membrane disks, which are the only elements of rod structure that appear to approach solid-state organization. These workers have proposed that the processes responsible for excitation occur at or very near the site of absorption of light and that excitation may then be conveyed to the receptor as a whole by some axial structure. NVald (i96o) has pointed out that all-trans retinal occurs in the visual process only as a transient intermediate which never accumulates. In contrast, there are instances in which virtually all the vitamin A of the eye is in the form of t i-cis retinal, as in the eye of the lobster. It appears that light is involved only in the first step of the rhodopsin cycle: the isomerization of z t-cis retinal to the all-trans form when the visual pigment is converted to the corresponding lumi-pigment. Visual excitation probably depends upon the initial steps in the cycle, that is, the formation of the lumi- or at most the meta- pigments (Wald, i96o). In view of the papers discussed above concerning the physical- chemical properties of polyene molecules, we tentatively suggest that an important feature of the isomerization of i r-cis retinal to all-trans retinal is that this process yields a molecule with a high electron mobility. It is known that deviation from a planar configuration, as in the twisted and bent chain of t z-cis retinal, sharply decreases the extent of overlapping of the p atomic orbitals and thus the degree of conjugation (Ferguson, 1963). Hence, i r-cis retinal would be expected to behave much less effectively as an electron transfer agent than the all-trans retinal that is formed by the action of light on rhodopsin. This property of r i-cis retinal might possibly be respon- sible for the widespread distribution of the compound in visual systems. Further, it is conceivable that, in this particular hindered cis isomer, the electrons of vitamin A are sufficiently localized and immobile to enable retinal to be stored in the membranes of the eye in a relatively inactive form, Thus isomerization of t i-cis retinal by light may not only be associated with the initiation of the visual impulse, but may also produce a molecular pathway for the transmission of electrons associated with the visual impulse. 1
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oto- was duc- SooA) T sbWtion ,OV made dcttrrent; Cherry photo- Gibbons ~ gttunt for ~ $tch as 6e double- ~~ approach ~ ~sportsible It excitation t. pcCss only mt instances *l,tsinthe trhodopsin val pigment $ly depends #tbemeta- kphysical- i important ;hia process -iou from a `pdecreases onjugation rauch less ned by the be respon- irther, it is min A are abranes of light may produce a t impulse. { I Vitamin A and cell fiinction (3) Olfaction and taste 443 Briggs & Duncan (196 1) have investigated the nature of compounds located on the interface between the epithelial cells and the air cavity of the nose. Bovine olfactory tissue in z% digitonin solution was extracted with ether and, although the extract gave absorption peaks at a variety of wave-lengths in the visible and ultraviolet regions, it was deduced that free x- and J3-carotene and vitamin A were present. Further experiments indicated the presence of protein-bound carotenoids from which the chromophore could be removed by acetone. It was suggested that protein-bound carotenoids of the olfactory epithelium are the receptors of energy from olfactant molecules entering the nasal cavity. Clinical data from 56 patients having uncompli- cated anosmia, who were treated by intramuscular injections of large doses of vitamin A, showed partial or complete recovery of olfaction in fifty cases. These observations indicate that olfaction might perhaps be included with photosynthesis and vision as yet another process in which the electron mobility that characterizes carotenoid molecules may be utilized in a process of energy reception and transfer. It is interesting that vitamin A deficiency causes abnormall taste responses in rats (Bernard, Halpern & Iiare, ig6z). A marked decrease in rejection of quinine and selection of sodium chloride solutions was observed in advanced stages of deficiency, while administration of vitamin A resulted in rapid recovery of sodium chloride selection but not of quinine rejection. Although Bernard et alL suggest that this indicates that vitamin A may have a direct effect on the functioning of the taste cells, it would seem that the effect might well be an indirect one. (4) Respiration cornpared with plrotosyntlresis Possible relationships between photosynthesis and vision have already been men- tioned. Another, and perhaps as fundamental similarity to photosynthesis is to be found in respiration. Respiration and photosynthesis are functionally alike in that both synthesize adenosine triphosphate (ATP), which is essential as a source of energy in the reactions of cellular biochemistry. Furthermore, the mechanism by which ATP is formed are similar, since both involve linked sequences of reactions in which the energy of electrons is tapped off sequentially. Structurally, there is a resemblance between the highly-ordered layering of membranes that is characteristic of chloroplasts and the complex membranous structures of mitochondria: these being the two organelles where the processes of photosynthesis and respiration respectively are located. Lehninger (ig6i), who has drawn the parallel between photosynthesis and respira- tion in detail, has remarked that the electron carriers in the respiratory chain bear many chemical similarities to those of •the corresponding chain in photosynthc~is. For example, riboflavin and cytochromc structures occur in both systems. To pur."ue this point, the similarities in composition also extend, in part, to lipids; thus, phos- pholipids occur in chloroplast lamellac («'intermans, ig6o), and they are not only present, but are an essential feature of the electron transfer chain in mitochondria (Fleischer, Brierley, hlouwen S: Slautterback, 1962; Brierley, Merola & Fleischer, ' • s8-s
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`=g6z). In mitochondria, coenzyme Q (ubiquinone) is closely concerned in electron transfer (Hatefi, ig63), while chloroplasts contain large amounts of plastoquinone, a ,Compound related to coenzyme Q. Evidence for the function of plastoquinone in photosynthetic electron transport in chloroplasts is similar to that for the involvement of coenzyme Q in mitochondrial electron transfer (Crane, 1961). x-Tocopherol occurs in chloroplast lamellae (Park & Biggins, 1964), and also in mitochondria, where it may possibly be a component of the cytochrome c reductase portion of the terminal respiratory chain and function as an electron carrier or a binding agent (Vas ington, Reichard & Nason, ig6o). The involvement of a-tocopherol in the respiratory chain, however, is considered unproven by other workers (cf. Pitt & gNiorton, i96z). According to Pitt & Morton (ig62) a majority of workers regard the physiological importance of vitamin K in oxidations and phosphorylations as slight = or non-existent in animals, although this may not be so in some micro-organisms. I ~,\iartius (196 1) has, however, postulated a role for vitamin K in electron transfer and i- oxidative phosphorylation while, as far as plants are concerned, vitamin Ki is present in chloroplasts (Park & Biggins, 1964) and may be concerned in photosynthetic phos- phorylation phor3lation (see Isler & Wiss, igsg). At this juncture, it may not be irrelevant to refer to the distribution of some of these compounds in bacteria. Coenzyme Q seems to participate in bacterial photosynthesis (Clayton, 1962); Green & Mascarenhas (1964) have suggested that all the coenzyme Q of Rliodospirilhrm rubrum is located in the cell membrane. According to Goodwin (1963), all photosynthetic bacteria synthesize carotenoids, which togetherwith bacterio- chlorophyll, are concentrated exclusively in the chromatophores. In a study of the } non-photosynthetic bacteria Escherichia coli and 1tlicrococcus lysodeikticus, Bishop & - King (ig63) observed coenzyme Q and vitamin K to be located in the cell membrane which, as these workers remark, is believed to be the site of mitochondria-like func- tions in such organisms. Carotenoids are apparently not present in all non-photo- synthetic s3•nthetic bacteria, but it is perhaps significant that according to Goodwin (1963) they are invariably absent from anaerobic organisms. Mathews & Sistrom (1959) found that carotenoid pigments, cytochrome oxidase, phospholipid, DPNH oxidase and ; succinfc dehydrogenase are associated with the cell envelope of Sarcina lutea. Since they showed also that the carotenoids are not in the cell wall, these workers concluded that the pigment is associated with the cell membranc.. There seems to be relatively little information, on carotenoids or compounds in the vitamin A series in relation to mitochondria. Basford & Green (1959) have described the preparation and properties of a soluble lipoprotein (coenzyme Q lipoprotein) from the succinic dehydrogenase complex of ox-heart mitochdndria. Approximately 6% of this lipoprotein is neutral lipid, which contains coenzyme Q, cholesterol, glycerides and an 'unknown carotenoid-like component with absorption maxima at 430, 455 and 4,$s mlC'. Retiny1 acetate, vitamin K1, and a-tocopherol, as well as coenzyme Q, are rendered `ticater-soluble' when added to, an aqueous solution of the lipoprotein. ~ Green (1959) has referred, in passing, to the possibility that carotenoids may eventually t prove to have a place in the electron transport system of mitochondria. Bouman & Slater (1956) mentioned carotenoids as occurring in a preparation of heart mitochon- ~~.. _
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445 drial fragments. The presence of a carotenoid-like compound in ox-heart mitochon- dria that has absorption maxima at 426, 45 i and 478 m1c in petroleum cther has been confirmed in this laboratory (Berger, Dingle & Lucy, unpubl.). As far as analyses for the presence of vitamin A in mitochondria are concerned, the position is far from satisfactory. Subccllular fractions of liver have been analysed on several occasions but, as liver is the main site of stored vitamin A in the body, the problems of contamination make it difficult to conclude whether vitamin A found in liver mitochondria is functional or merely being stored. Collins (1952) found that approximately ao% of the total vitamin A of a single rat liver was in the mitochondrial fraction, and a similar figure was obtained by Powell & Krause (1953). Krinsky & Ganguly (1953) investigated the subcellular distribution of retinyl ester and retinol in rat liver. The ester was found to be concentrated in the cream fraction of the liver homogenate, while retinol was present in this fraction and in the supernatant and microsomal fractions. These workers considered that the quantity of vitamin A in the mitochondrial fraction was of an order of magnitude consistent with contamination. They therefore concluded that the mitochondrial, and also the nuclear, fractions were essentially free of vitamin A, and they suggested that retiny 1 ester and retinol are associated with different proteins during storage within the liver cell. It should perhaps by noted that the stated object of these experiments was to elucidate the actual . site of storage of both forms of vitamin A. Quantities of retinol that would justifiably appear negligible in relation to the total amount of vitamin A stored in the liver might, nevertheless, be highly important from the point of view of the structure and function of the membranous elements of the liver cell. It has been suggested in this eonnexion that, if the vitamin is normally present in mitochondrial membranes, absence of the vitamin in deficiency conditions, as well as the presence of excess in hypervitaminosis, might be expected to cause distortion of mitochondrial structure (Lucy et at. 1963). Bouman & Slater (1956) found less than o-oi pmole of vitamin A per g. of protein in a preparation of heart mitochondrial fragments. However, ab- normally swollen mitochondria have been obtained from the tissues of animals suffering from vitamin A deficiency (DeLuca, personal comm.), and Sheldon & Zetterqvist (1955) have described changes in mitochondrial morphology in the keratinizing epi- thelia of vitamin A-deficient mice. (5) Vitamin A and electron transfer It appears from the data discussed in the preceding paragraphs that there is no conclusive evidence for the functional presence of vitamin A in mitochondria. At this point, discussion on vitamin A in relation to electron transfer might perhaps be dismissed as sterile. As yet, however, nothing is known of the fundamental bio- chemical mode of action of the vitamin in many of its physiological functions and" the idea that retinol, or a metabolic product, may be concerned in electron transfer is not so unlikely as not to deserve serious consideration. Thus, we have seen that the chemical structure of vitamin A and carotenoid compounds is such that it might well facilitate the movement of electrons throuah a lipid environment. "Purthcrmore, a number of workers believe that carotenes arc involved in electron transfer in phvto- s8-1
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' 4 46 J. T. DI\GLE A:,'D J. A. Lucy s,ynthesis. Then, too, we have seen that t photos3-nth his in chloror la,ts and'resp'rration d l d The spatial relationships of the components of the respiratory chain are of great interest at the present time (for a recent review see Ernster & Lee, 1964). Chance, sn attempt to see how vitamin A or carotenoids might possibly fit into the picture. " ~,nd function of mitochondria and lipoprotein membranes will be briefly discussed, in I Tn the next three paragraphs certain aspects of current concepts on the structure ~tinol interacts strongly with lipoprotein membranes and membrane lipids, whereas Closely related but ph}'siologicalI}- inactive compounds do not. both organelles; there is also some evidence that vitamin A may be concerned in ~piration (see Section III). Finally, the work discussed in Section II shows that• t at certain ipi s are common to n tnitochondria nave many stmi ar eatures, an *sctive centre of the next haematin in the electron transfer chain. Fernandez-IbZorin 1 -Whieh would enable an electron-bearing haematin to come into contact with the phorylation. The individual haem-containing components making up the oxysome are thought to be able to interact with one another by rotational or trar._lational movements Estabrook & Lee (1963) have introduced the concept of the `oti-ysome' as a macro- molecular assembly having the capabilities of electron transfer and oiidative phos- ~ Oda, Blair & Green (1964) think that there is no evidence of molecular collisions i hypothesis that the repeating particle (elementary particle), which is associated with the cristae and the inner membrane of ox-heart mitochondria in electron micrographs the electron transfer chain. These workers have published data in support of their between adjacent proteins witnm tne 'soua-state matnx' ot ttte complexes making up of how electrons move within each complex, has referred to a suagestion by Bock & 'Criddle that the proteins make contact with one another b,v means of swinging groups particle) containing a complete electron transfer chain that can be isolated from these 3nitochondria. Green & Fleischer (1963) have suggested that the oxidation-reduction groups of the chain are so orientated as to abut into or fit «-ithin the phospholipid that is associated with each of the complexes. More recently, Green (1964), in a discussion of negatively stained preparations, is identical with a particulate unit (electron transfer I f atoms, mounted on the respective proteins by flexible arms, that transfer and , ACcept electrons within the lipid moiety of each complex- ; Lehninger (rg62) has suggested that from 25 to 40°,0 of the membrane protein of mitochondria could be made up of respiratory chain and energy-coupling enzymes arranged in 'solid-state' assemblies more or less evenly distributed in the membrane, ' multi-enzyme systems rather than metabolically inert `skins' (cf. Ball & Barrnett, and he considers mitochondrial membranes as complex fabrics of regularly spaced 1957, and the cytoskeleton of Peters (1956) discussed above). These ideas have been illustrated by the diagram which is reproduced in Text-fig. 2 (Lehninger, rg6i). Recent studies made with the electron microscope- on the structure and assembly f negatively stained macromolecular lipid complexes indicate that it is apparently unnecessary to consider structures containing a high proportion of phospholipids lely in terms of bimolecular leaflets. Apparently lamellar structures, that might >tsstaeiation of small globular micelles of lipid (Lucy & Glaucrt, 1964). In the ~ be regarded as bimolecular leaflets may, in certain circumstances be formed by the. O r O Co 4~* NO N
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Vitamin A and cell funution 447 light of these observations, the possible relevance of globular,znicelles of phospholipid (4o'So A in diameter) to the structure and function of membranes has been considered and some of the properties of a theoretical micellar model for the lipids of bio- logical membranes have been discussed (Lucy, iq64a). It was suggested that the adaptability and versatility of biological membranes may result from the utilization of both bimolecular leaflets and globular micelles of lipid molecules, and from equilibria between the two configurations. Attention was drawn to the likelihood that, in the .light of Haydon & Taylor's discussion (1963), the presence of non-phospholipid molecules such as vitamin A in the membrane would decrease the stability of the bimolecular lipid leaflet and encourage the formation of globular micelles. In addition, it was pointed out that a micellar structure for certain areas of membranes might provide a means of enabling enzyme molecules to be structurally incorporated into the plane of the lipid layer. Thus, one or a number of lipid micelles in an organized array Text-fig. m. A hypothetically schematic representation of a respiratory assembly in the mito-, chondrial membrane. The assembly is indicated by blacl circles; it includes three sets of coup- ling enzymes arranged laterally to account for oxidative phosphorylation of adenosine diphos- hate. The open circles indicate the structural protein of the membrane. Reproduced with per- mission from Lehninger (zg6i). could be replaced by globular protein molecules (Text-fig. 3). Relatively large pro- tein molecules could be inserted provided that the local disorder in the array of globular lipid units, and the consequent increase in size of the aqueous pores between the units, did not become a limiting factor in the functioning of the membrane. It was thought that an array of globular enzymes and globular micelles of lipid would be particularly suitable for membranes whose primary function is to provide a locus for complex enzyme reactions (cf. Mitchell & Moyle, 1958). In view of the various considerations that have been discussed in the present article, it seems possible that vitamin A, or a metabolite retaining the essential con- jugated system and mobile electrons of thf.carotenoids, might transfer electrons from one site to another in membranes while remaining embedded in the Fhos pholipid. In mitochondrial membranes, this mechanism might be additional to the rotational and translational movement of proteins in the oxysome hyPothcsis, and an alternative to the proposal that swinging groups of atoms, mounted on the proteins by flexible
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44$ J. T. DINGLE AND J. A. Lucy arms, transfer electrons through the lipid. It might also be suggested that movement of electrons, via vitamin A or a derivative, through the interior of globular micelles of phospholipid could provide an electronic connexion between different enzyme mole- cules of the electron transfer chain (Text-fig. 3). These enzymes would be an integral part of the membrane, but they would comprise part of a lipid layer and not part of a protein layer as suggested by Lehninger (ig6t) (see Text-fig. 2). At this point it should perhaps be emphasized that, although the possibility of retinol or related molecules participating in electron transfer in a hypothetical micellar-type membrane Text-fig. 3. A surface view of the lipid moiety in a snicellar model for a biological membrane (reproduced, with additions, from Lucy, zg64a). The globular micelles of lipid (unshaded) are shown in hexagonal close packing, but such a structure r ould probably be flexible and the indi- vidual micelles in continuous random movement. The lipid cores of these nucelles would have diameters of about 4o A; water-filled pores (P), approximately 4 A in radius, are shoa.•n be- tween the micelles. The shaded units represent globular proteins with enzymic or hormonal properties that have replaced the lipid micelles at certain points in the plane of the lattice. Both isolated enz}7ne molecules and an organized array of several different enzymes are illustrated. Two phospholipid micelles are supposed to contain molecules of vitamin A or of a metabolite. The arrows between these two micelles and their adjacent enzyzne molecules illustrate diagram- matically, for a micellar-type membrane, ihe suggestion that electrons may flow between indi- vidual protein carriers of the electron transfer chain via the conjugated double bonds of mole- cules which have the carotenoid skeleton. Similarly electrons may be transferred from a protein component to a lipid component, such as coenzyme Q. has been discussed in some detail, these molecules might equally well transfer electrons through membrane lipids that are arranged quite differently, for example, in bi- molecular leaflets or in other structures. In conclusion, it may be pointed out that, even if vitamin A or a related compound is concerned in mitochondrial oxidative phosphorylation, it does not necessarily follow that it is involved in the main pathway of electron transfer. Indeed, in view of the observations of Nishimura & Chance (ig63) (seeSection IV (r)) that carotenoids may be involved in the flow of electrons in the photosynthesis and respiration of certain . t . . i N cn © ~ 0 0o %0 4, .~,
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compound rily follow view of the noids may of certain Vitamin A and cell function . 449 .bacteria but apparently are not on the main pathway of elettron transfer, it would seem that similar considerations could apply to animal mitochfndria. The increased oxidation of Krebs' cycle intermediates that occurs in vitamin A deficiency (see Sectiorl III) might then be interpreted to mean that vitamin A is concerned iits a regulatory side-path that may be available, as required, for rapidly shunting electrons away from the main chain leading to oxygen; deficiency of the vitamin may then lead initially to an uncontrolled increase in oxidation. V. 'ACTIVE FORMS' OF VITAMIN A , The possibility that retinol itself might not be the active form of vitamin A has interested investigators for many years. Studies on this problem made prior to 1957 ~ were discussed by Moore (1957) who concluded that, taken as a«•hole, evidence for hidden forms of vitamin A may be considered fragmentary and inconclusive. Never- theless, Moore presented reasons for thinking that an intermediary product is con- ~d cerned in the mechanism of action of the vitamin; for example, when single doses of the vitamin are administered to depleted rats, it seems necessary for at least ioo i.v. to disappear before measurable storage begins. Furthermore, in some of the sites that are particularly vulnerable to the effects of vitamin A deficiency, such as mucous mem- branes and the urinary bladder, it does not seem possible to demonstrate the presence of the vitamin by chemical means even during normal health. Some recent observa- tions on 'active forms' of vitamin A will now be discussed. , The possibility that retinoic acid may be the active form of vitamin A in many of its non-visual functions has recently been considered. This compound is not stored in the liver, a fact first noted by Arens & van Dorp (ig.}6). Retinoic acid, which apparently cannot be reduced to retinol or retinal, is biologically active in many respects but it is ' inactive in vision (Dowling & Wald, ig6ob) and is unable to support the reproductive processes of rats (Thompson, Howell & Pitt, 1964). If retinoic acid is the systemically active form of vitamin A, then the rat must rely on the continuous oxidation of its stores of vitamin A for a supply of retinoic acid. However, Olson (1964) has recently ' concluded that, although retinoic acid seems to be a metabolically active intermediate, as yet this compound has not been conclusively shown to be a product of retinol meta- bolism. Jurkowitz (i96z) has discussed technical considerations concerning the • estimation of retinoic acid which indicate that the presence of relatively large quantities may pass undetected in certain circumstances. Working on the assumption that a compound formed from retirioic acid may be the active form of the vitamin, a number of investigators have studied the mctabolism of f the acid. Apparently several active metabolitics have been obtained and Olson (1964) has tabulated the preliminary information available; he has drawn attention to the ' fact that most of the isolated metabolites are more polar than rctinoic acid, but are , extractable from acidic solution by ether. Yagishita et al. (i964) have recently reported experiments with an acidic metabolite ('compound 5') that is formed frum retinoic acid or retinol by loss of at least the•carbon atom no. ta. The weights of retinol- -deficient animals returned to normal after feeding with S.pg. of compound 5, or its -M-00M.MM"
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450 J. T. DINGLE AND J. A. Lucy ` ester, for three days. It would seem reasonable to suppose that any active form of vitamin A should, however, be at least as active as retinol itself and it might be pre- dicted that considerably smaller quantities of compound 5would restore growth rates in deficient rats if this compound is indeed the active form of the vitamin. Zile & DeLuca (1964) have also recently isolated and fractionated metabolites from the livers of rats fed with labelled retinoic acid. Four major radioactive components were obtained, all of which were chromatographically different from retinol, retinoic acid and retinal. One of the components was biologically active and possessed growth- promoting action comparable to that of retinol. To conclude this Section, reference might perhaps be made to our own studies on hypervitaminosis in model systems that have already been discussed in Section II. If membranes are indeed one of the physiological sites of action of the vitamin as we have suggested, then, as far as the primary interaction of the vitamin with membranes is concerned, it would seem that retinol itself may normally be the active form of the vitamin. This is indicated by the relatively specific ability of retinol to penetrate the red cell membrane and to interact with a lecithin-cholesteroll monolayer at an air- water interface. Retinol may not remain unchanged, however, once it has entered the lipoprotein framework of the membranes. By analogy with photosynthesis and bac- terial respiration, the 'active form' might be expected to be a carotene; retinol might then be regarded as the vehicle by which the membranes of animal cells receive the carbtenoid skeleton, which, unlike plant and bacterial cells, they cannot synthesize for themselves. In contrast to derivatives of retinol or retinoic acid, carotene mole- cules might be sufficiently long to facilitate transfer of electrons from one side of a bi- molecular leaflet of phospholipid to the other, or through a globular micelle of phos- pholipid (cf. Text-fig. 2) without the carotene molecules having to be arranged in a back-to-back fashion. However, although carotenoids appear to be present in ox-heart mitochondria, there is apparently no evidence for. a back reaction by which vitamin A may be converted to a cartotenoid. For example, in analyses of mitochondrial lipids made in this laboratory, it has been found that although small quantities of caro- tenoids are normally present in rat-liver mitochondria, they are absent when rats are maintained on a carotene-free diet supplemented with vitamin A (Berger, Dingle & Lucy, unpublished). Nevertheless, if the vitamin does function in electron transfer associated with animal lipoprotein membranes, retinol may be converted in mem- branes to a molecule or molecules as yet unknown that can best fulfil this function. It is conceivable that the active molecules may differ slightly from one tissue to another but that they may all have the same conjugated system of unsaturated double bonds. VI. CONCLUSIONS It may be concluded, from the experiments `on hypervitaminosis A discussed in Section II, that some, but by no means all, of the effects of excess of vitamin A spring initially from the ability of the compound to interact with the plasma and intracellular membranes of cells. It seems likely that interactions occur between the phospholipids of membranes and molecules of retinol, and that some of the damaging actions of hypervitaminosis A result from the subsequent oxidation of the double bonds of . t 0 r%) tn 0 F~ 0 ~ ~ ~ %o a+
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tom of be Pre- rates 'e & the were acid wth- ranes of the ie the air- 's&red the ilttnd bac- jVd might £ #Ceive the t qttthesize out mole- 10e of a bi- * of phos- in a -heart 'tritamin A iWlipids .. ~_. a_oi' caro- are are , Dingle & -cOtransfer a in mem- 'unction. It to another *bonds. isTussed in iA A spring 'tracellular spholipids mctions of = bonds of I , i c Vitamin A and ce11 function 6 :. retinol and the co-oxidation of membrane lipids, that may occur when too many molecules of retinol penetrate membranes. Similarities in the relationships between molecular structure and function are seen when the actions of excess vitamin A on membrane systems are compared with prevention of deficiency. This indicates that membranes may also be concerned in the action of the vitamin under physiological conditions. At the present time, however, there is not sufficient biochemical knowledge about the mechanisms whereby vitamin A prevents deficiency symptoms in the animal to • conclude whether or not lipoprotein membranes are involved. A number of suggestions have been made by us and by other workers of mechanisms that involve changes in the permeability or stability of membranes, by which vitamin A may control cellular functions. In the present article consideration has been given to a further possibility: that vitamin A, or a compound derived from vitamin A by metabolic reactions occurring in the membranes of mitochondria or other organelles, may function as an electron transfer agent. The degree of attention accorded here to membranes -: in general and to mitochondrial membranes and electron transfer in particular does not, however, imply that we consider membranes to be the only site of action of vitamin A. The several objections that can justifiably be raised concerning the thesis that vitamin 3, or a metabolite, may function in lipoprotein membranes have not been discussed at length here, though many have been mentioned in passing. The most significant objection would seem to be that much of the experimental evidence for the surface-active properties, and hence the 'membrane-active' properties of the vitamin, come from studies on hypervitaminosis. Extrapolation from these studies to the mode of action in alleviating deficiency must naturally be made with due caution. llore- over, although there is evidence that the vitamin plays a part in mitochondrial oxidations, this action is little understood. l~evertheless, we hope that our various suggestions concerning possible relationships between the physico-chemical pro- perties of the vitamin and its physiological functions may serve to stimulate further research. vII. SUMMARY z. Both deficiency and excess of vitamin A produce many diverse pathological changes. Lipoprotein membranes have recently been found to be concerned in a number of the actions of excess of the vitamin. This article is devoted principally to a discussion of the interactions and possible functions of vitamin A.vithin membranes, both in hypervitaminosis and under physiological conditions. 2. Excess of vitamin A. (a) As a result of its amphipathic molecular structure, retinol is highly surface active. (b) The initial action of excess of rctinol on crythrocytes is an expansion of the cell membrane; this is followed by haemolysis unless the cells are kept cold, or inhibitors, such as vitamin E, are present. (e) Addition of retinol to fibroblasts growing in vitro causes degranulation anci 2501080497
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swelling of the endoplasmic reticulum, swelling of Golgi vacuoles and mitochondria, and the formation of cytolysomes. (d) Isolated mitochondria from certain tissues also swell.in the presence of retinol. This swelling is apparently not dependent on respiration, but is inhibited by vitamin E. (e) Lysosomal enzymes are released by excess of retinol, both in vi,:o and in vitro. A number of the effects of excess vitamin A on connective tissues mav be ascribed to this action-; these effects are inhibited by hydrocortisone, which stabilizes lysosomes, but are not significantly inhibited by vitamin E. (f) Closely related, but physiologically inactive, derivatives of retinol are relatively inactive towards membranes; membranes might therefore also be concerned in the physiological functions of retinol. (g) Vitamin A has recently been shown to have marked effects on the membranes of certain bacteria and viruses. g. Deficiency of vitamin A. (a) The diminished synthesis of mucopolysaccharide observed in deficiency may result from an interference with the synthesis of 'active sulphate' but, in view of conflicting observations, this cannot be considered as proven. (b) Suggestions have been made that lipoprotein membranes may be concerned in the actions of vitamin A on both mucopolysaccharide and steroid biosynthesis. (c) The action of the vitamin in biological oxidations is far from clear. Recently it has been found that deficiency increases, and excess of vitamin A decreases, the oxida- tion of succinate by homogenates of rat liver. (d) Organ culture experiments indicate that vitamin A acts directly on mouse pros- tate glands to prevent the squamous changes that are characteristic of deficiency. 4. A conjugated chain of alternating single and double bonds is a chemical feature common to vitamin A and the carotenoids. (a) Such a chain is characterized by a high electron mobility; this has been studied both theoretically and experimentally in relation to electron transfer in photo- synthesis, to ion transport, and to super-conduction and semi-conduction in biological systems. (b) The role of vitamin A in vision may be an evolutionary development of the function of the carotenoids in photosynthesis. In the eye, retinal is intimately con- cerned with the structure and function of lipoprotein membranes. We have suggested that changes in electron mobility may be functionally associated with the isomerization of i i-cis retinal by light. (c) Vitamin A, or carotenoids, may be concerned in the receptor systems of olfaction and taste. (d) Photosynthesis in chloroplasts has many structural, chemical and functional features in common with respiration in mitochondria. Carotenoids are found in ox-heart mitochondria but the presence of vitamin A is uncertain. (e) We have suggested that vitamin A or a metabolite retaining the conjugated chain may be present under physiological conditions in the lipids of biological membranes and that, by virtue of its mobile electrons, it may function in mitochon- drial electron transport. :
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hitamin A and cell furution ` 4q-,; ~. Atctive forms of vitamin A. - (a) Recent work indicates that retinoic acid, or a metabolite of retinoic acid, may be the active form. It has not been established conclusively, however, that retinoic acid is normally formed in vivo from retinol. (b) -6is conceivable that if vitamin A is concerned in electron transfer, the vitamin ? may penetrate lipoprotein membranes as retinol and that it may subsequently be converted to a functional derivative. In this way, the membranes of animal cells may receive 'the carotenoid skeleton which, unlike plant and bacterial cells, they cannot td in M. gtcently it the osida- gpust pros- Acknq_ &jW feature 9~ has been AG in photo- ki biological =t of the z9lfely con- ;e suggested merization ; of olfaction I functional 'e found in conjugated f biological t mitochon- = synthesize for themselves. ~~ We are most grateful to Dame Honor Fell, F.R.S., Sir Rudolph•Peters, F.R.S., and Miss Audrey M. Glauert for reading the manuscript of this article and for their helpful suggestions. We thank %Sr R. A. Parker and Mr I. Armond for preparing the plates and figures, and Mrs M. Wright for her careful ; typing. : x .. .. VIII. REFERENCES i -' ALI, S. Y. (Ig64). The degradation of cartilage matrix by an intracellular protease. Biochem. ,3! 93, 6I r-i$. ' A.%tis, S. R.; SwANsoN, W. J. & HAStxIS, P. L. (I955). Biochemical studies on vitamin A. XIV. Bio- potencies of geometric isomers of vitamin A acetate in the rat. J. am. Chem. Soc. 77, 4t34'6• ; ArzE:%s, J. F. & vaN Doar, D. A. (1946). Activity of 'vitamin A-acid' in the rat. Nature, Lond., i58, i 6u-3. AYnELo•rrE, M. B. (Iq63a). The effects of vitamin A and citral on epithelial differentation in vitro. t I. The chick tracheal epithelium. 1. Embryol. exp. 1llorph. u, z79-9I. AYDELOT'rE, M. B. (1963b). The effects of vitamin A and citral on epithelial differentation in vitro. ~ 2. The chick oesophageal and corneal epithelia and epidermis. ,y. EmbryolL exp. 3lorph. zz, 62I-35. BALL, E. G. & BARRNErI', R. J. (I957). An integrated morphological and biochemical study of a purified preparation of the succinate and DPNH oxidase system. ,7: biophys. biochem. Cyto1. 3, to23-36. ; BANGIIA.\s, A.'D., DINGLE, J. T. & Lucy, J. A. (1964). Studies on the mode of action of 'excess of vitamin A. 9. Penetration of lipid monolayers by compounds in the vitamin A series. Biochenr. ,7. 90, t33-4o. I3ARRNErr, R. J. & BALL, E. G. (i96o). Metabolic and ultrastructural changes induced in adipose tissue by insulin. ,7. biophys. biockem. Cytol. 8, 83-ior. ' BASFoxD, R. E. & GREvr, D. E. (I959). Studies on the terminal electron transport system. XXI. On the properties of a soluble lipoprotein dissociated from the succinic dehydrogenase comples.• Biochim. ~ biophys. Acta, 33, I85-94• ~ BERNARD, R. A., HALPERN, B. P. & KARE, M. R. (I96I). Effect of vitamin A deficiency on taste. Proc. Soc. exp. Biol., N.Y., io8, 784-6. 1 BISxoP, D. H. L. & KING, H. K. (Ig6t). Ubiquinone and vitamin K in bacteria. 2. Intracellular distribution in Escherid:ia coli and Micrococcus lysodeikticus. Biochem. .7. $$, 550-.}. BLouea, H. A. (I963). A molecular approach to tetratogenesis: effect of vitamin A on influenza virus 1 in ovo. Nature, Lond., 199, 33-5• BourrlAN, J. & SLATER, E. C. (I956). Tocopherol content of heart-muscle preparations. Nature, Lond., =77,ii8I-2. BRIERLEY, G. P., MEROLA, A. J. & FLEISCxER, S. (t962). Studies of the electron-transfer system. KLIX. Sites of phospholipid involvement in the electron transfer chain. Biochim. biophys. Acta, 64, 2I8-s8. BRIGGS, M. H. & DuNOA.v, R. B. (I96I). Odour receptors. Nature, Larrd., 191, I3IO-II. BRowx, P. K., GlnsoNSS, I. R. & WALD, G. (I963). The visual cells and visual pigment of the mud puppy, Necturus. ,3'. Cell Biol. 19, 79-to6. BRowlv, R. G., BurroN, G. M. & S.nTtI, J. T. (I963). A hypothesis for aconunon mode of action of the fat soluble vitamins, A, D and E. I- theoret. Biol. 5, 489-92. CALVIN, AI. (I95g). Energy reception and transfer in photosynthesis. In Biophysical science-a study program, pp. I47-56, ed. J. L. Oncley et al. New York. CsANCE, B.,,ESTADROOK, R. W. & LEE, C. P. (1963). Electron transport in the oxysome. Scierrce, 140, 379-80• . . • CHAPAfAN, D. & CHERRY, R. J. (1964). Current pulses in fl-carotene. Nature, Loud., 203, 641-2. • ' CHArrFLt., J. B. .C-. GREVtLLE, G. D. (t958). Dependence of mitochondrial swellings on oxidizubte" substrates. Arature, Lond., t82, 8t3-i4.
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In The structure of the eye, pp. 85-99, ed. G. K. Smelser. New York. Dovt'LitiG, J. E. & WALD, G. (x96oa). The role of vitanun A acid. Yitorn. ~`''-, Horm. r8, Sxg 4x. Do«zt:cG, J. E. & WALD, G. (xg6ob). The biological function of.dtamin A acid. Proc. Nat. acad. Sci., Wash., 46, 587-608. DuvE, C. nE (t959). Lysosomes-a new group of cytoplasmic particles. In Subccllular particla, pp. xz8-S9, ed. T. Hayashi. New York. DuvE, C. DE,'kVArnAux, R. & Wiso, M. (x96t). Effects of fat-soluble compounds on tysosomes in titro. Biocher.r. Plrarmacol. 8, 30 ER.Ys'rElt, L. & LEE, C: P. (xg64). Biological oxidoreductions. Ann. Rev. Biochem. 33, 7Z9-88• FELL, H. B. (tg6x). The influence of IlydrocortisaiIe on the metaplastic action of vitamin A on the epidermis of embryonic chicken skin in orgaasn culture. J. Embryol. exp. Morph. xo, 389-409. FELL, H. B. (I964a). Some factors in the regulation of cell physiology in skeletal tissues. In Bone biodynamics, pp. t89--207, ed. H. M. Frost. Boston. FELL, H. B. (tg646). The role of organ cultures in the studies of vitamins and hormones. Vitam. & Horm. (in the press). FELL, H. B. & DIxGLE, J. T. (x963). Studies on the mode of action of excess of vitamin A. 6. Lysosomal protease and the degradation of cartilage matrix. Bioclunl.,f : 87. 403-8. FELL, H. B., DINGLE, J. T. & WEaa, M. (I962). Studies on the mode of action of excess of vitamin A. 4. The specificity of the effect on embryonic chick-limb cartilage in culture and on isolated rat-liver Iysosomes. Biochem. ,7: 83, 63-9. . FE.L, H. B. & MrLLAxaY, E. (r952). The effect of hypetaitaminosis A on embryonic limb bones cultured in,roitro. ,7. Physiol. 116, 320-49. FeLL, H. B. & Titatwtes, L. (x96o). Comparison of the effects of papain and vitamin A on cartilaga II. The effects on organ cultures of embryonic skeletal tissue. ,3: exp. Med. zxx, 7x9-44. FELL, H. B. & Txo.rras, L. (xq6 t). The influence of hydrocortisone on the action of excess vitamin A on limb bone rudiments in culture. 1. exp. Med. 114, 343"6Z. FERGusoN, L. N. (1963). The modern structural theory of organic chemistry. . Englewoods Cliffs, New Jersey. FERN,LVDEZ-MottAIV, H., ODA, T., BLAIR, P. V. & GREEN, D. E. (x964). 'A macromolecular repeating unit of mitochondrial structure and function. Correlated electron microscopic and biochemicai studies of isolated mitochondria and submitochondrial particles of beef heart muscle. Y. cell Bfol. 22, 63-too. FITTON JACKSON, S. & FELL, H. B. (t963). Epidermal fine structure in embryonic chicken skin during atypical differentiation induced by vitamin A in culture. h?eul Biu1. 7, 394-419. FLSisCIIER, S., BRIE•RISY, G., KLOUWE,r, H. & SLAUT'I'eRaACx, D. B. (t96z). Studies of the electron transfer system. XLVII. The role of phospholipids in electron transfer. ,7. biol. Cllem. 237, 3264-72. GLAuEtRT, A. M., DANIEL, M. R., Lucy, J. A. & DtxGLE, J. T. (t963). 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1 Vitamin A and cell functiom. 455 GooowiN, T. W. (1963). The biosynthesis of vitamins and related compodnds. London. GREEN, D. E. (I959)• Mitochondrial structure and function. In Subcellular particks, ed. T. Hayaahi, PP• 84-103. New York. GREh:N, D. E. (t964). The mitochondrion. Sci. Amer. 210 (t), 63-74. GREEN, D. E. & FLEISCIiER, S. (1963). Role of lipids in mitochondrial electron transfer and oxidative phosphorylation. In Biochemical problems of lipids, pp. 323-45, cd. A. C. Frazer. Amsterdam. GREEN, D. E. & LESTER, R. L. (t9S9). Role of lipids in the mitochondrial electron transport system. Fed. Proc. :8, g87-tooo. GREEN, J., DIPLOCIt, A. T., BttNYAN, J., EDwtS, E. E. &'%IcHAI.E, D. (t96t). Ubiquinone (coenzyme Q) and the function of vitamin E. i\ature. Lond., i9o, 3I8-z5- GREENBERG, S. M., CALBERT, C. E., PINCIi.kRD, J. H., DECEL, H. J. & ZEICHtiiEiSTER, L. (1940- Stereo- .• chemical configuration and provitamin A activity. IY. Comparison of all-trans-y-carotene and•pro- y-carotene with all-trans-f.3-carotene in the chick. Arch. Biochem. 24, 3 t-9. GREITIE, A. F. & MASCARENHAS, J. P. (t964). Coenzyme Q; intracellular distribution in Rhodospirillum rubrum. Science, i4.4, I455-6• HALKERSTON, I. D. K., EICHHORN, J. & Hechter, 0. (t959). TPNH requirement for cholesterol side chain cleavage in adrenal cortex. Arch. Biochem. 85, 287-9. HATEfI, Y. (t963). Coenzyme Q (ubiquinone). Adt•. Enzymol. 25, 275-328. HAYDON, D. A. & TAYLOR, J. (1963). The stability and properties of bimolecular lipid leaflets in aqueous• solutions. ,7. theoret. Biol. 4, 28I-96. HuNTEtt, F. E., GESIcxI, J. M., HoFFsro;, P. E., Scorr, A. & ZS'EL.-rEtti, J. (1963). Swelling and lysis of rat liver mitochondria induced by Fe } ions. ,3: biol. Chem. 238, 8z8-35. HYONo, A., KURIYAMA, S., Tsuji, K. & HosoY.s, Y. (t962). Monolayer film of rhodopsin at the air- water interface. Nature, Land., z43, 679-80. Ist.z:R, O. & Wiss, O. (1959). Chemistry and biochenistry of the K vitamins. Vitam. & Sorm. z7, 53-yo. JAHN, T. L. (t962). A theory of electronic conduction through membranes, and of active transport of ions, based on Redox transmembrane potentials. ,7. theoret. Biol. 2, 129-38. JuRKOtviTZ, L. (1962). Determination of vitamin A acid in human plasma after oral administration. Arch. Biochem. 98, 337-4t. KutI:ER, H. E. (tg6o).. Electron microscopic study of the phagocytosis process in lung. .7. biophys. biochem. Cytol. 7, 357-66. KtxstcY, S. C. (1963). Comparative responses of mammalian erythrocytes and microbial protoplasts to polyene antibiotics and vitamin A. Arch. Biochem. xoz, i8o-8. Ktu:vKsY, N. I. & GANGULY, J. (t953). Intracellular distribution of vitamin A ester and vitamin A alcohol in rat liver. Y. biol. Chem. 202, 227-32. LASNITZKI, I. (tg62). Hypovitaminosis :A in the mouse prostate gland cultured in chemically defined medium. Exp. Cell Res. 28, 4o-5t. LEIININCER, A. L. (tg6t). How cells transform energy. Sci. Am. 205 (3), 63173- LEHNINGER, A. L. (1962). Water uptake and extrusion by mitochondria in relation to oxidative•phos- phorylation. Physiol. Rev. 42, 467-$t7. LEHNINGER, A. L. & RAY, B. L. (t957). Oxidation-reduction state of rat-liver mitochondria and the action of thyroxine. Biochim. biophys. Acta, 26, 643-4. LEIistINCER, A. L., RAY, B. L. & SCHNEInER, M. (t959). The swelling of rat liver mitochondria by thyroxine and its reversal. J7. biophys. biochem. Cyto1. g, 97-to8. LEjEUNE, N., THINES-SE1tPOUX, D. & HERs, H. G. (t963). Tissue fractionation studies. t6. Intra- cellular distribution and properties of a-glucosidases in rat liver. Biochem. _7. 86, I6-2t. LtTTLS, W. A. (1964). Possibility of synthesizing an organic superconductor. Physical Rev., 134, A t416-24. Lucy, J. A. (t964a). Globular lipid micelles and cell membranes. ,7 theoret. Biol. 7, 360-73. LueY, J. A. (tg64b). Membrane permeability and the control of cellular function. National Cancer Institute Monograph, no. 13, 93-to7. LucY, J. A. & DINGLE J. T. (tg62). Vitamin A and membrane s.`stcros. 2. Membrane stability and protein-vitamin A-lipid interactions. Bioclu•m. ,7. 84, 76 ~ P. Lucy, J. A. & DINGLE, J. T. (t964). Fat-soluble vitamins and biological membranes. Nature, Load., 204, 256-60. Lucy, j. A., DINGLE, J.'h. & FELL; Ii. B. (tg6t). Studies on the mode of action of vit:unin A. 2. A possible role of intracellular proteases in the degradation of cartilage matrix. Biochcrn..7. 79, 500-s• Lucy, J. A. & Gl-.%UERT, A. A'I. (t964). Structure and assembly of macromolccular lipid cotnplexes composed of globular micclles. I. mol. 13io1. 8, 727-48. Lucy, J. A., Lt.TscomnE, M. & DINGLE, J. T. (r963). Studies on the mode of action of excess of vitamin A. 8. Mitochondrial swelling. B{ochern. J7. 89, 4 t9-r. 2501089501
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M+Rrtrs, C. (ig6i). Recent inves^cations on the chemistry and function of vitamin K. CIBA Founda- tion Symp. on Quinones in electr..~n transport, pp. 312-26, ed. G. E. W. Wolstenholme and C. M. O'Connor. London. ~1+rxEtvs, M. M. & StsrROm, W. R. (1g59). Intracellular location of carotenoid pigments and some respiratory enzymes in Sarcina iaaa. 7. Bact. 78, 778-87. 'h1tt.ks, N. A. (tgi;). Vitamins A a.d carotenes. II. Chemistry and industrial preparation. • In The vitamins, vol. 1, pp. 4-5S, ed. W. H. Sebrcll and R. S. Harris. New York. MITCHELL, P. &\foNz.E, J. (s9.Z). Group-translocation: a consequence of enzyme catalysed group- 1IooRE, T. (rg6z). The bioche.-nic:1 mode of action of vitamin A. Nutritio et Dieta, 3, i-2z. MoRE-m, A. & WoLF, G. (t96t). Vitamin A and mucopolysaccharide biosynthesis in colon homo- genates. Biochim. biophys. Acta, 53, 263J72. lfoxroNz, R. A. (i961). L'biquir.onea (coenzymes Q), ubichromenols, and related substances. Vitam. & Horm. ig, r-42. Moxrox, R. A. & Ptrr, G. A. J. (rg57). Visual pigments. Fortch. Chem. organ. Naturstofje, 14, 244-316. MoRTOX, R A., S{L+H, M. K. & STCBss, A. L. (i947)• Retinenes andvitamin A=. Nature, Lond., z59, 744• Nisxtatmk, 1•1. & Cx.LNcE, B. (1953). Studies on the electron transfer systems in photosynthetic bacteria. x. The light-induced absorption-spectrum changes and the effect of phenylmercuric acetate. Biochint. biophys. 3cta, 66, z-i6. NovZi:oFF, A. B. (t963). Lysosom;!--z in the physiology and pathology of cells: contributions of staining methods. Ciba Found. S}"por.'1.-: on lysosomes, pp. 36-77, ed. A. V. S. de Reuck and M. P. Cameron. London. OLso,t, J. A. (Ig64). The biosynthesis and metabolism•of carotenoids and retinol (vitamin A). ,7. lipid Res. g, 28r-gg. P.+.xb, R. B. & Bicot,s, J. (r96{). Quantasome: size and composition. Science, i44, ioog-ii. PAStEfwAic, C. A., HrasrxRiFS, S. K. & PiRiE, A. (1963). The activation of sulphate by extracts of cornea and colonic mucosa -o:n normal and vitamin A-deficient animals. Biochem. ,3: 86, 3824. P+u2, J. & PEaRso.-,, E. S. (ig6o). The action of insulin on the metabolism of cell cultures. Y. Endocrin. 2i, 287-94- PErERS, R. A. (t956). Hormones ard the cytoskeleton. Nature, Lond., 177,426. Ptrr, G. A. J. & Z1oRrox, R A.. (ig6o). Cis-trans isomers of retinene in visual processes. Sytnp. biochem. Soc. sg, 67-89. " Ptrr, G. A. J. &MoRTo.-,, R. A. (t962). Fat-soluble vitamins. Annu. Rev. Biochem. 31, 49I-5t4• PLarr, J. R. (t95g). Carotene dot:o:-acceptor complexes in photosynthesis. Science, x2g, 372-4- PotvELL, L. T. & Iix~ rsE, R. F. (19 g3). Vitamin A distribution in the rat liver cell. Arch. Biochem. 44, zoz-6. PtTLL.m.~.v, B. & PL'LLNL,.v, A. (z963). Quantum biochemistry. New York. RAO, K. S. & GA.\GL2Y, J. (r964). Studies on metabolism of vitamin A. 6. The effect of vitamin A deficiency on the activation of si:lphate and its transfer to p-nitrophenol in rat liver. Biochem.,3C go, io4-9.. Rto, K. S., 5.asrttY, P. S. & G~uzGtZ.Y, J. (1963). Studies on metabolism of vitamin A. 2. Enzymic synthesis and hydrolysis of pheno?ic sulphates in vitamin A-deficient rats. Biochem.,3: 87, 3r2-r7. RAY, A. & S.+Dm:, D. P. (r 9:9). Oxidative processes in hypervitaminosis A in albino rats. Am. 1. Physiol. i96, r274-6. Raztv, S. & ARGA]Lsoc, \1. (i963). Lt sis of mycoplasma, bacterial protoplasts, spheroplasts and L-forms by various agents. _7. gen. alicrc:iol. 30, 155-72. REDFF-aR.v, E. R(1956). The eLe,.-t of vitamin A deficiency on the activity of the liver succinoxidase system in the rat. Biochem. _7. 6;, 39P. RoELS, O. A., TROr•r, M. & GrY=.. A. (1964). Vitamin A deficiency and acid hydrolases: fl-glycero- phosphate phosphatase in rat livrr. Biochem.,y. 93, 23e-25e• RosENuERC, B. (t959). Photocor.d-:c-:ion and cis-trans isomerism in 6-carotene. .7. chem. Physics, 31, 238-46. RosE.vuERc, B., ORLLNDo, R. A. & ORLA_NDO, J. M. (t961). Photoconduction and semiconduction in dried receptors of sheep eyes. Arch. Biochem. 93, 395-8. SEBRELL, W. H. & HARRrs, R. S. (t-)c,). The vitamins, vol. t. New York. SEa•aRD, C. R. & HorE, E. L. (1964). Respiratory decline in vitamin A-deficient rat liver homogcnates. transfer. Yature, Lond., 192, 37s-3. 1\fooRE, T. (i957)• I~7itamin A. Ar=terdam. Fed. Proc. 23 (2), 293•
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_Wlone `~ Ilomo- ~ Vitam. totgos, =4. ,t, j er+E, :59, owy't,thetic ;~entuic ytOf ftain/ng •, p.Osneron- ,t A)• J. Iipid rtt. , extracts of Jnt. 1: 86, j; Endocrin. nes. Symp• .9I-5I4• ;7z-4. 4iodiem- 44, vitamin A 90. z. Enzymic 7, 3I2-i7. -s. A:n. ,3: :d L-forms cinoxidase .8-glycero- Jrysics, 3z, 3uction in aogenatea. Vitamin A and cell function " 457 6 SHELDON, M. & ZETTERQVtST, H. (1955). Experimentally induced changcs in mitochondrial morphology: vitamin A deficiency. Exp. Cell Res. 10, 225-8. SLEDGE, C. B. & DINGLE, J. T. (t965). Activation of lysomes by oxygen. 11'ature, Lond., 205, I4o-t. TAPLEY, D. F. & CoaPER, C. (t956). Effect of thyroxine on the swelling of mitochondria isolated from various tissues of the rat. Nature, Lond., 178, Itt9. THO:KPSON, J. N., HOWELL, J. McC. & PITr, G. A. J. (t964). Vitamin A and reproduction in rata. Proc. roy. Soc. B, z59- 5IO-35• VAN DYKE, R. A., WOLF, G. & JOHNSON, B. C. (ig6o). The function of vitamin A in adrenal steroid production. Biochem. biophys. Res. Comm. 3, I23-6- VARANDA.*II, P. T., WOLF, G. & JOHNSON, B. C. (ig6o). Function of vitamin A in the synthesis of 3'-phosphoadenosine-5'-phosphosulphate. Biochem. biophys. Res. Comm. 3, 97-too. VASt:vcTON, F. D., REICHARD, S.1-T. & NASON, A. (ig6o). Biochemistry of vitamin E. Litam. & Horm. i8. 43-87- VICKERIIAN, Ii. (z 962). Patterns of cellular organization in Limax amoebae. Exp. Cell Res. 26, 497-5t9• VIGNAts, P. V. (I957). Action de la vitamine A sur la transhydrogdnase et la TPNH cytochrome c reductase. Exp. Cell Res. z3, 4r4-i6. WALD, G. (1943). The photoreceptor functions in the carotenoids and vitamin A. Vitam. & Horm. z, 195-227. WALD, G. (ig6o). The visual functions of the vitamins A. Yitam. & Horm, i8, 4t7-3o. WA_NG, D. Y., SLATER, F. & DAxTNALL, H. J. A. (t963). Swelling properties of retinal mitochondria. Biochem. _7. 86, 5 P-6 P. WEISS, L. & DINGLE, J. T. (I963). Lysosome activation in relation to connective tissue disease. Annu. rizeum. Dis. 23, 57-b3 • WEISSSLI_N,, G. & DIt.eLE, J. T. (tg6t). Release of lysosomal protease by ultra violet irradiation and inhibition by hydrocortisone. Exp. Cell Res. 25, 2o7-to. WEISSKAN, G. & THo%ias, L. (t963). Studies on lysosomes. 2. Effect of cortisone on release of acid hydrolases from a large granule fraction of rabbit liver induced by excess of vitamin A. J. clin. Invest. 42, 66t-9. WEtssaii.v, G. & THOai,is, L. (I964). The effect of corticosteroids upon connective tissue and lysosomes. Rec. Progr. Horm. Res. 20, 2t5-45• WEITZEL, G., FxEI'zDoRFF, A. & HELLER, S. (t952). Grenzfl5chenuntersuchungen an Verbindurigen der Vitamin A-Gruppe. Hoppe-Seyl. Z. 290, 32-47. WILI-MER, E. N. (I96I). Steroids and cell surfaces. Biol. Rev. 36, 368-98. WINTER.~SA.VS, J. F. G. M. (ig6o). Concentrations of phosphatides and glycolipids in leaves and chioro- plasts. Biochim. biophys. Acta, 44, 49-54• WoJczAK & LEILVIVGER, A. L. (tg6z). Formation and disappearance of an endogenous uncoupling factor during swelling and contraction of mitochondria. Biochim. biophys. acta, 51, 442-56. WOLF, G. (r962). Some thoughts on the metabolic role of vitamin A. A'utr. Rev. 20, I6t-3. WOLF, G., BERGAN, J. G. & St,xDaRIsAN, P. R. (t963). Vitamin A and mucopolysaccharide biosyn- thesis by cell-free particle suspensions. Biochim. biophys. Acta, 69, 524-32. WOLF, G., LANE, M. D. & JOHNSON, B. C. (t957). Studies on the function of vitamin A in metabolism. ,9! biol. Chern. 225, 995-1008. WOLF, G. & VARA.'YDANi, P. T. (ig6o). Studies on the function of vitamin A in mucopolysaccharide biosynthesis. Biochim. bioplzys. Acta, 43, 5ot-t2• WOLF, G., VARANDANI, P. T; & JoHNSON, B. C. (t96I). Vitamin A and mucopolysaccharide synthesiz- ing enzymes. Biochim. biophys. Acta, 46, 59-67. WOLF, G., WACLE, S. R., vA.v DvKE, R. A. & JotLNsoN, B. C. (I958). The function of vitamin A in metabolism. II. Vitamin A and adrenocortical hormones. J7. biol. Chem. 230, 979-go. YAGISHITA, K., SUNDARESAN, P. R. & WoLF, G. (t.964). A biologically active metabolite of vitamin A and vitamin A acid. Nature, Lond., 203, 4IO-t2. ZECHMEISTER, L. (ig6o). Cis-trans isomeric carotenoid pigments. Fortschr. Chem. org. Naturst. i8, 223-349• ZECII\tE-ISTER, L. (t962). Cis-trans isomerie earotenoids, t•itamins A and arylpolyeius. Vienna. ZILE, M. & DELucA, H. F. (I964). A biologically actic•e'metabotite of vitamin A acid. Fed. Proc. 23 (2), 294• trJ
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458 J. T. DINGLE AND J. A. LL:CY EXPLANATION OF PLATES PLATE I Electron micrographs of thin sections of rabbit erythrocytes; (a), (b) and (c) fro:a Glaucrt et al. (z963), (d) and (c) from Lucy & Dingle (t964). xc. ig,ooo. (a) A normal red cell suspended in saline. (b) Surface indentations and vacuole formation resulting from treatment with retinol (to ag./rnl.) for several minutes at 2g° C- (c) Red cell ghosts formed on incubation of the cells with retinol (tg pg.;tr1.) for 15 min. at 37° C- (d) Indentations produced in the cells 30 sec. after addition to retinol and DL-a-toco- pheryl acetate (go fig./mI.), at zs' C. (e) An intact cell containing vacuoles after i5 min. at 37° C. with retinol (I3 ftg.fmL) and DL-a- tocopheryl acetate (50 /ig./m1.). (a), (b) and (c) are reprinted by permission of the Rockefeller Institute Press, (d) and (e) were originally published in ATature, Lmrd., and are reprQduced with permission. PLATE 2 Electron micrographs of thin sections of cultured rat dermal fibroblasts (unpublished work by Daniel, Dingle, Glauert & Lucy). x C. 30,000. (a) A cell from a control culture. Fine fibrous material (F) is present lying parallel to the surface membrane. The cisternae of the endoplasmic reticulum (ER) are filled.trita dense material and a mitochondrion (.\i) shows signs of slight damage. (b) A cell from a control culture; an extensive Golgi zone (G) and microrubu!es (arrow) are present in the cytoplasm of two adjacent cells. (c) A cell after 12 hr. incubation with retinol (33 pg./ml.) at 37` C. The resicles in the Golgi zone (G) and the mitochondria (\1) are swollen. The,cisternae of the endoplasmic rericulum (ER) have collapsed. (d) A cell after 9 hr. treatment with retinol (33 pg./ml.) at 37° C. The cytopla.s= contains cytolysomes (CY). and a lipid droplet (L) and microtubules (double arrow). An apparendy helical aggregate of n'bosomes (arrow) is present. IX. ADDENDUM Oxidation of retinol. Dispersion of retinol in colloidal form is apparently a pre- requisite for its action on the membrane of the red cell (Lucy, 1965). Furthermore, retinol in colloidal dispersion is very rapidly oxidized by molecular oxygen. Autoxida- tion of retinol in saline solution is associated with the development of spectroscopic fine structure; maxima at 310, 325 and 340 m;w suggest the formation of a retro-com- pdund that has only four conjugated double bonds. The long-chain isoprenoid com- pounds tt-tocopheryI acetate, vitamin Kl, phytol and squalene not only inhibit rapid haemolysis by retinol but also inhibit autoxidation of retinol in saline. It has therefore been suggested that these substances may influence the structure of colloidal particles of retinol and that they might perhaps form 'intra-micellar' inclusion compounds with retinol. These observations are thought to lend support to the thesis that haemolysis by retinol in vitro may result from co-oxidation of membrane lipids. Jungahwala & Cama (1965) have described the preparation and properties of a num- ber of 5,6-monoepoxy and 5,8-monoepoxy derivatives of vitamin A. The metabolism and biological potency in the rat of certain of these compounds has also been investi- gated (Lakshmanan, Jungalwala & Cama, i965). It is of considerable interest that 5,6-monoepoxyretinal was observed to have a biological potency of ioS % that of a1l- trans retinyl acetate. Furthermore, a new visual pigment which had an 'absorption +:,.a;
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.ady a pre- pft}lermorC, Autoxida- Ictroscopic , retro-com- tnoid com- Nbit rapid tstherefore 1aI particles aounds with : haemolysis :s of a num- metabolism )cen invcsti- interest that a that of all- i'absorption Biological Reviews, Vol. 40, No. 3 DINGLI: aNu LUCY . (b) -. J4)- ia ~.. ~ . .. , it f ~ . 1 :.~1+. ;A.}' . ~ ~l}r~• S~y ) • A'- .t ` •~ •S j ... ~i tit. 1 T a °` ~ ` :i ] ~ l J. .-_-• r;s r~. an. q• r ~r (C)
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Vitamin A and cell function a 459 maximum at 480 mµ was isolated, in addition to natural rhodopsin, from the retinas of rats maintained on 5,6-monoepoxyretinal. Only traces of vitamin A were detected in the livers of rats maintained on 4,5-monoepot-}-retinal. Lakshmanan et ulL consider that these trace amounts were not likely to have arisen from the epoxy compound, and they suggest that the biological activity of the epoxide is not mediated through the intermediate formation of vitamin A. Their findings indicate, as the investigators point out, that the four conjugated double bonds in the chain of vitamin A, rather than the 8-ionone ring, may be the essential molecular criterion for biological activity. It would appear to be significant, however, that the introduction of epoxide groups into ,8-carotene greatly reduces activity. Thus, fl-carotene 5,6,5',6'-diepoxide has earlier been observed to have only i$ % of the activity of /3-carotene (Isler & Zeller, I957)• To develop the idea put forward by Zechmeister (ig6o) (see Section II) that biological activity may depend on whether or not a molecule has the right shape to fit onto an enzyme surface, it may be suggested that a virtually unmodified /3-ionone ring might perhaps be essential for the enzymic conversion of carotenoid precursors to vitamin A but not for the subsequent metabolism of vitamin A to its 'active' form. _ Lysosomal enzymes. Hsu & Tappel (1965) have investigated the action of retinol on the activity of arylsulphatase and fl-glucuronidase. These enzymes, which are generally thought to be of lysosomal origin, were obtained from colon, small intestine, kidney and liver of the rat. Retinol (too frg.!mg. protein) was found to activate ary1- sulphatase and to inhibit /3-glucuronidase; retinyl acetate was less effective than retinol whilst retinoic acid was without activity. Hsu & Tappel suggest that retinol may function as a coenzyme for arylsulphatase. However, in view of the high concentra- tion of retinol employed in these experiments and the known affinity of the vitamin for proteins (cf. Lucy & Dingle, 1964), the possibility that retinol caused non-specific changes in the structures of the enzymes studied should perhaps be considered. Viruses and tissue culture. The cellular transformation in cultured epithelial tissues induced by excess of vitamin A (Fell & 'Mellanby, 1953) has been used by Huang & Bang (ig64) in a study of virus multiplication. They found that conversion in vitro of chick embryonic epidermis to mucous epithelium by to-.}o pg./ml. of retinyl acetate increased the yield of influenza virus, but decreased the production of vaccinia virus. This change appeared not to be due to vitamin A per se but to be related to the change in differentiation of the tissue supporting the v irtis. Isoprenoid synthesis. Relationships between vitamin A and isoprenoid synthesis in the rat have been investigated by Diplock, Green & Bunyan (1965). It was confirmed that the concentration of coenzyme Q in liver is markedly greater in deficient rats than in control animals (cf. Morton, zg6i); it was also observed that heavy sypplementation with retinyl palmitate depresses the concentration of coenzyme Q. However, Diplock et al. found no consistent effect of vitamin A deficiency, or of vitamin A dosage; on the incorporation of mevalonate into cholesterol or into squalene, and there appeared to be no evidence for a specific effect of vitamin A on isoprcnoid synthesis at the meta- bolic level. Their findings therefore do not confirm earlier observations which indi- cated that in deficiency an increased -synthes is of coenzyme O occurs in rats which is associated with a block in the synthesis of cholesterol from squalene (Gloor S Wi•s, 2501p$9507
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460 J. T. DINGLE AND J. A. Lucy 1959a, b; Phillips, 1961). Joshi, Jayaraman & Ramasarma (1965) have observed that in short-term experiments (2 hr.) the incorporation of mevalonate into cocnzyme Q in the livers of deficient rats is less than normal. Since orally-administered, radio- active coenzyme Q was deposited in the livers of both normal and deficient animals but subsequently lost only from the livers of the normal animals, these workers have suggested that the increased concentrations observed in deficiency may result from reduced degradation of coenzyme Q in the liver. Retinoic acid. The testes of the cock, unlike those of the rat, develop and remain healthv when the methyl ester of retinoic acid is used as a source of vitamin A (Thomp- son, Hon-ell, Pitt & Houghton, 1965). Nevertheless, methyl retinoate is not only un- able to support vision in poultry but it is also unable to sustain normal embryonic development, although it allows the adult hen to maintain egg production. Thompson et al. have concluded that the failure of chick embryos to develop normally in eggs. from hens receiving methyl retinoate orally is a manifestation of vitamin A deficiency, since injection of retinol, retinyl acetate or retinal into the eggs before incubation stimulated normal development and, in some instances, enabled normal chicks to be hatched. This work therefore appears to add one more specialized and differentiating system to the list of those in which retinoic acid is unable to fulfil completely the functions normally met by retinol, retinyl esters or retinal. Dunagin, Meadows & Olson (i965) have investigated the metabolism of retinoic acid in rat bile and have tentatively identified the major metabolite as retinoyl fl-gluc- uronide. They have suggested that the rapid conjugation of retinoic acid with gluc- uronic acid in the liver, its excretion in conjugated form in the bile and its partial re- absorption from the intestine may throw light on the rapid disappearance of free retinoic acid from tissues and also on the presence of water-soluble metabolites of retinoic acid in the liver and intestine. One of the arguments in favour of retinoic acid being the active form of vitamin A rests on the failure of liver to store retinoic acid and on the swift disappearance of the compound from the body. If conjugation of retinoic acid with J3-glucuronic acid and its subsequent excretion is responsible for both these observations, we would suggest that there is considerably less reason to believe that retinoic acid is nearer to the active form of vitamin A than retinol. REFERENCES DtpLoctc, A. T., GxEEx, J. & BuxvAN, J. (1965). Vitamin A and isoprenoid synthesis in the rat. Bio- thrmJ•95, 138-43• Dtr\,AGtv, P. E., MEADO%vs, E. H. & OLSON, J. A. (i963). Retinoyl beta-glucuronic acid: a majormeta- bolite of vitamin A in rat bile. Science, i48, 86-7. FELL, H. B. & MELLAAIIiY, E. (i953)• Metaplasia produced in cultures of chick ectoderm by high vita- min A. .7. Physiol. i r9, 470-88. GLOOR, U. &Wtss, O. (t959a). On the biosynthesis of ubiquinone (5o). Arch. biochem. 83,216-22. GLooR, U. & Wiss, O. (t959b). Influence of vitamin A deficiency on the biosynthesis of cholesterol, squalene and ubiquinonc. Biochem. biophys. Res. Comm. i, ISz-i. IfvANC, J. S. & BANG, F. B. (i964). The susceptibility of chick embryo skin organ cultures to influenza virus following excess vitamin A. Y. exp. A1cd. 120, i 29-48. • Hsu, L. & TAPPEt, A. L. (t965). Effect of vitamin A on the activity of arylsulfatase and /1-glucuronidase of rat tissues. Biochim. biophys. Acta, zoz, 173-20. IsLER, O. & ZELLER, P. (1957). Total synthesis of carotenoids. Iitan:iru E=3 Hormones, IS, 31-71. i I t I I iZ
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- BFo- metsi- AICtlronldase » 5t_7i. i=~ • . Vitamin A and cell function 461 JosHt, V. C., JA, ARA-M.av, J. & RAMASAR.aA, T. (I965). Some observations on the metabolism of co- enzyme Q and ubichsornenol in rat tissues. Biochem. biophys. Res. Comm. s8, I o8-I4. JUNCALWALA, F. B. Ec CA..tA, H. R. (sg65). Preparation and properties of 5,6-monocpoxyvitamin A acetate, 5,6-monoepon-vitarnin A alcohol, 5,6-monoepoxyvitamin A aldehyde and their corresponding 5,8-monoepoa-y(n:ranoid) compounds. Biochenz.,3'. 9S, I7-z6. LAKSHMANA,-, tl. R, Jt.'\cAL«•A[a, F. B. & CAMA, H. R. (t965). Metabolism and biological potency of 5,6-monoepon-vitnamin A aldehyde in the rat. Biochem.,7. g5, 27-34. Lucy, J. A. (I965). autosidation of retinol, and its inhibition, in an aqueous environment. Biothem.,7. (in the press). PHILLIPS, W. E. J. (tg6t). Vitamin A deficiency and isoprenoid metabolism. Can. ,7. Biochem. 39, 855-6r. THOHIPSON, J. N., HovvELL, J. 'McC., Pirr, G. A. J. & HoucHroN, C. I. (zg65). Biological activity of retinoic acid ester in the domestic fowl: production of vitamin A deficiency in the early chick embryo. Nature, Lond., 205, Ioo6-7.
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t . O 1968 by Academle Press Ine. ). tTLTRASTRUC't1JRE RESEARCH 22, 206-230 (1968) Hyperplasia and Cornification of the Transitional Epithelium in the Vitamin A-Deficient Rat Changes in Fine Structure of the Cells R. M. Htct:s • The Bland-Sutton Institute, The Middlesex Hospital Medical Scfrool, London. It! 1, England Received October 2, 1967, and in rerised form December 11, 1967 The bladders of rats maintained on a%itamin A-deficient diet develop patches of cornified epithelium that are histologically comparable to epidermis, and also areas in which there is a stratum granulosum but no stratum corneum. In the electron microscope, a marked similarity was cbserved bet«een the fine. structure of cornified bladder epithelium and that of skin. Prickle celis, with a marked fibril desmosome r,-lationsnip were found. and the dense l:eratohyalin- tike areas in the stratum granulosum were associated with fibrils and/or ribo- somes. There is kr.ou n to be a close association between ribosomes and kerato- hyalin in both skin and oral mucous membrane, and a similar association was observed here in the cornified areas of bladder epithelium. In the noncornified areas, no organized cytoplasmic fibrils, prickle cells, or stratum corneum +vas found, but the stratum- granutosum contained dense keratohyalin-like material. This material was not associated with fibrils and was apparently derived from aggregates of morphologically altered ribosomes. It is suggested that the formation of fibrils and the formation of keratohyatin may be mutually independeat but normally coincident processes in keratinizing squ3mous epithelia. It is also suggested that in these vitamin A-deficient ani- mals, the fibrils in the cornified bladder develop from the cytoplasmic filaments seen in normal transitional epitheli3l cells. and that keratohyalin is formed by enzymatic degradation of aggregated ribosomes. These results are related to an earlier suggestion that molecular keratin is probably synthesized in normal transitionat epithelium, even though it is not usually cornified. Wolbach and Howe (39) were the first to observe gross cornification of the urinary bladder of the vitamin A-deficient rat, and more recently the histology of this con- dition has been described (8). These earlier observations have now been confirmed, and birefringent fibrils were observed in the cornified patches of bladder epithelium (16). The response of the bladder in most animals maintained on a vitamin A-defi- cient diet for 8-20 weeks is characterized by the appearance of large keratohyalin-like
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In the present study the changes in the bladder epitheliuni of rats maintained on a vitamin A-deficient diet are described, and the observations are discussed in the con- text of an earlier suggestion (14) that normal bladder epithelium should be regarded as a keratinizing tissue, i.e., one in which molecular keratin is synthesized, which is not normally cornitied. granules in the superficial cells, which, however, are not always overlaid by a stratum corneum (16). These bladder granules, like skin keratohyalin, apparently contain ribonucleic acid, calcium, and/or magnesium, but no disulfides, and their-reactions to a variety of cytochemical stains were the same in the presence or absence of a stra- tum corneum (16). It has been suggested that skin keratohyalin represents strongly staining regions of the fibrils, where they are permeated by an interfibrillar matrix (6). The observation that keratohyalin-like granules can occur in the absence of a stratum corneum composed of birefringent fibrils (16) is therefore of some interest, and in the present study the fine structure of these granules was investigated. The similarity between cornifying epithelia produced by lack of vitamin A and mamma- lian epidermis was remarked upon by Bern (2), and it is therefore also of interest to .. compare the general fine structural changes in vitamin-deficient bladder epithelium _ with published accounts of the fine structure of skin (6, 7, 19, 28, 33). the urinary of this con- confirmed, epithelium min A-defi- hyalin-like ~ FINE STRUCTURE OF VITAMIN A-DEFICIENT BLADDER 207 MATERIALS AND METHODS Animals. Weanling (3 week old) rats of the Wistar strain weighing about 50 g at the start of the experiments were used both for these and the previously reported light micro- scope findings (16). . Diet. A pelleted vitamin A- and ascorbic acid-deficient diet was obtained from Nutritional Biochemicals Corporation, (Cleveland, Ohio). Vitamin A waass obtained as 'Ro-A-Vit,' which contains 150.000 IU of vitamin A per milliliter, from Roche Products Ltd. (Welwyn Garden City, England). • Treatment of animals. Thirty weanlings were fed ad libitum with the pelleted vitamin A- deficient test diet. Supplementary ascorbic acid was given in the drinking water as a 20 mg °. solution. After 3 weeks on this diet, each animal received orally a weekly supplement of 75 IU of vitamin A. This dosage is known to prolong survival without allowing storage of vitamin A in the liver (8). Twenty other weanlings were kept as controls housed in adjacent „ cages, but fed ad libitum with a complete stock diet. Vitamin A-deficient and parallel control ~ animals were killed together at weekly intervals between 4 and 20 weeks after the start of r; the experim:nt. Chemicals. Epikote 812 was obtained from George T. Gurr Ltd. (London. S.W. 6., Eng- land). land). All other chemicals used were standard laboratory reagents of the highest purity : available. - Electron microscopy. One ureter and parts or the bladders from each animal were sec- ,} tioned into small pieces under cold, 1°a (w/v) osmium tetroxide, buffered with phosphate (24). The tissue was dehydrated, embedded in Epikote 8 12, and prepared for examination ` in a Siemens Elmiskop I by the methods described previously (1S).
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208 B basal epithelial cel! O a basal attachment plate C blood capillary E endothelia, cell b basement membrane F fibroblast C centt'iole H histiocyte d desmosome. I intermediate cell e endoplasmic reticulum L bladder lumen f fibril :yM matrix of desquamated layer k keratohyalin-tike body S squamous epithelial cell m mitochondria Z bundle ceU r ribosome t filaments r ftuifotm vacuole All figures. except Fig. 16, are electron micrographs of thin sections of rat bladder epithelfal cells which have been fixed in osmium tetroxide and embedded in Epon. Fig. 16 is a light micrograph of £pon-embedded tissue. The tissue shown in Fig. 17 was contrast stained with uranyl acetate. That shown in all other electron micrographs was contrast stained with both uranyl and lead salts. FtG. 1. Part of a basall cell from a cornified area of vitamin A-cleficient rat bladder. The basal cell is separated from the subepithelial tissue by a basement membrane (b). Basal attachment plates (a) can be seen on the cell membrane, and the cytoplasm contains mitochondria (m), a few cisternae of endo- plasmic reticulum (e), many ribosomes (r), and filaments (t), some of which are arranged in bundles to form fibrils(A - 49,000. FtG. 2. The field shows a longitudinal section through the intermediate, prickle cell layer of a corni- fled area of bladder epithelium. The cells at the bottom of this field are in contact with basal cells, . not shown here. The edges of the prickle cells are extended into lon processes those of adjacent cells in the extracellular space. All the prickle el s contain ahich interdigitate net- work of fibrils ; j) which extend into the cell proesses, where each fibril tetminates n a desmosome (d). Also przseat in this field is a small process of a histiocyte (H) which has less dense cytoplasm than the prickh cells. x 11,000. !:i ,I
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G a 0 a ® a ~,ILf;!wi a I : p k r R • v ~ Y t 4 0 0
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210 a. M. HICKS a Light microscopy. The other ureter and pieces of bladder from each animal were fixed for 6 hours in 4% glutaraldehyde buffered to pH 7.4 with 0.1 M sodium cacodylate (34), then washed for 16 hours in 0.25 Sf sucrose buffzred with 0.1 sodium cacodyfate to pH 7.4 and embedded in paraffin wax. Sections were stained by hematoxylin and eosin, and by 0.25% aqueous toluidine blue. For comparison, sections were cut from the Epon-embedded tissue which had been prepared for electron microscopy; these also were stained with totui- dine blue. RESULTS A very varied response of the bladder epithelium to vitamin A-deficiency has al- ready been reported (16). Cornified patches were found in one animal after only 8 weeks on the diet, and more regular cornification occurred after 15-20 weeks. The epithelial response in any one bladder also varied, from simple agranular hyperplasia, through noncornified granular hyperplasia. to the formation of a stratified squamous epithelium with an external cornified layer (16). In this report. the fine structure of an area of cornified epithelium is illustrated and described, and also that of an area of noncornified, granular hyperplasia. Areas of agranular hyperplasia were examined but are not illustrated here, as their fine structure ivas largely the same as that of normal transitional epithelium, which has been described elsewhere (13). The main differences were, first, that the stratum intermedium was composed of many layers instead of a single layer of cells, and second that the superficial cells, although limited by a thickened membrane, contained fewer fusiform vacuoles in the apical cytoplasm than did normal controls. All cells contained randomly arranged cytoplasmic fila- ments and ribosomes. Cornified bladder epitlrelittm The appearance of a cornified area of bladder epithelium is illustrated in Figs. 1-12. The cuboidal basal cells had a fine structure very similar to that of the basal cells in normal control animals. Thus there were many half-dzsmosomes, or basal attachment plates, on the cell membrane facing the basement membrane, and the cytoplasm contained numerous filaments, free ribosomes, mitochondria and a little endoplasmic reticulum (Fig. 1). Unlike the controls, the cytoplasmic filaments in these cells were aggregated to form fibrils, whose density, however, was not much Fto. 3. A longitudinal section through the stratum corneum and stratum granulosum of a cornified area of bladder epithelium. The stratum corneum. in the upper half of the field, is composed of flattened cells which contsin many filaments (t) and fibrils; j), and a few oval profiles (o) of unknown origin. No nuclei are present in this layer. The cells are` separated by a slightly dilated extracellular space containing a dense material which is particularly noticeable in, the regions of the desmosomes (arrows). The cells of the stratum granulosum in the lower half of the field contain dense masses of keratohyalin-like material (k). These are surrounded by ribosomes (r) and sometimes make contact (A) with the fibrils(j). -, 20,000.
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212 x. M. H:cxs a greater than that of the rest of the cytoplasm. At low magnification therefore these fibrils were not particularly conspicuous. The stratum intermedium from cornified areas of bladder epithelium differed con- siderably from that of controls. The cells were stellate and connected by numerous cell processes and desmosomes, and were similar in appearance to prickle cells in the epidermis (Figs. 2 and 4). The cytoplasmic filaments in these cells formed a network of fibrils that were conspicuously more dense than the surrounding cytoplasm, even at low ma_nification. The fibrils extended into the long cell processes where each terminated~in a desmosome (Figs. 2, 4, and 6). A few tight junctions were observed between the processes of adjacent cells (Fig. 7). The cytoplasm between the fibrils was packed with free ribosomes, and also contained mitochondria and a few mem- brane-bounded dense bodies similar to the membrane-coating granules found in skin (.9. 11, 12, 20, 27). At high magnification the dense bodies were seen to be laminated (Fig. 7), and each lamina had a unit structure as if composed of normal membrane. The upper cells of the stratum intermedium were in contact at their distal borders with the stratum granulosum. The stratum granulosum, which was two or three cells thick, is shown in longi- tudinal section below the stratum corneum in Fig. 3, and one cell from this layer is shown in cross section in Fig. 5. The cells of the stratum granulosum were charac- terized in the light microscope by the presence of granules with the same staining characteristics as keratohyalin (16), and in the electron microscope by large, irregu- larly shaped, dense areas in the cytoplasm. It can be seen in Fig. 5 that these dense areas were frequently found in close association with the cytoplasmic fibrils. They then had a similar appearance to skin keratohyalin (6, 19, 28, 33). This keratohyalin- like material appeared at higher magnification as a dense homogeneous mass in close contact with both fibrils and ribosomes (Fig. 8). However, the dense material was not always contiguous with the fibrils. Keratohyalin-like masses were frequently observed, surrounded by ribosomes but not associated with fibrils (Fig. 9), although it is possible that such contact was made beyond the plane of section. Dense keratohyalin-like masses were also seen within the nuclei of some cells in the stratum granulosum (Fig. 10). A little substructure within this material could be resolved (Figs. 8 and 11), but it did not appear to be in the form of filaments embedded in a dense matrix as descri- bedd for skin keratohyalin (7), but rather to be a fine homogeneous granular substance (Fig. 11). Fio. 4. This cross section through a prickle cell in a cornified area of bladder epithelium, shows the network of dense cytoplasmic fibrils (f), and the desmosomes (d) on adjacent cell processes. , 12.000. Fto. 5. This field shows a cross section through a cell from the stratum granulosum from the same area of bladder epithelium. Keratohyalin-like masses tk) can be seen in close association with the network of fibrils (f) around the nucleus (n). The cytoplasm between the fibrils is filled with ribo- somes. x 15,000. a% W ~ ~ V1 I
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214 R. M. HICKS Ia}•ers (Fig. 12). Desmosomes persisted in the stratum corneum, and the extracellular of the cornified cells were thicker and denser than those of the cells in the underlying but the origin or nature of such structures was not apparent. The limiting membranes corneum. An occasional, irregularly dense, oval profile persisted between the filaments ribosomes of the underlying stratum granuiosum could not be seen in the stratum were the masses of filaments (Fig. 12). The keratohyalin-like masses, and numerous disappeared. The only remaining cytoplasmic constituents which were identifiable of flattened cells from which the nuclei. mitochondria, and other cell organelles had to many cell layers thick. That illustrated in Fig. 3 had several, loosely packed layers The stratum corneum in cornified bladder epithelium varied from two, or three, most layers were completely separated or_desquamated from the rest (Fig. 3). vely more dilated toward the luminal surface of the cornified layer, and the outer- space between the areas of the desmosomes. The extracellular spaces were progressi- (Fig. 12). A little electron dense material also extended into the dilated extracellular space between their opposing plates was occupied by an electron opaque material As in the normal bladder, histiocytic macrophages were occasionally observed between the epithelial cells. Xoncornhed, granular bladder epithelium areas, such fibrils were few in number. groups of parallel filaments formed small fibrils, but, unlike the situation in cornified filaments, and a few cisternae of rough-surfaced endoplasmic reticulum. In some cells, were common and the cytoplasm contained numerous mitochondria, ribosomes. The basal cells did not differ markedly from those in the normal bladder. Mitoses able remnants of bundle cells persisted even in the desquamated mass (Fig. 14). bundle cells (13) lay free between the epithelial cells at all levels (Fig. 13). Recogniz- dant cytoplasm (Fig. 13). The intercellular spaces were a little dilated, and many An area of noncornified, granular hyperplasia of the bladder epithelium is ilius- trated in Figs. 13-19. The general appearance of this epithelium is shown in Figs. 13-15. As in the normal bladder, the subepithelial blood capillaries were closely applied to the base of the epithelium and were bounded by endothelial cells with abun- which lie at various angles to the plane of section. • 82,000. connecting two cell processes can also be seen. The cytoplasm contains ribosomes (r) and fibrils (f) normal m_mbrane. At the lower right of the field is a desmosome (d), and a tight junction (arrowed) a laminated matrix lies in the top left of the field. Each lamina has a unit structure similar to that of Fic. 7. A small area of prickle cell cytoplasm is shown here. A membrane-bounded dense body with of which each is composed. • 60.000. locatrd in this field are many cross-sectioned cytoplasmic fibrils ; j j, showing the individual filaments in this field. Desmosome junctions (d) may be seen on the membranes limiting the cell processes. Also Fte. 6. A number of adjacent prickle cell processes in cornifi=d bladder epithelium are illustrated t f
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6IS680T0SZ
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~ = w~,1~ : ,3' -~:i ~ 1G'-~r'K(~' ~ e ;}'e r' . s . s. ig*- Fte. 8. Cytoplasm of cells in the stratum granulosum of cornified bladder epithelium. The kerato- hyalin-like material (k) shown here is in close contact both with ribosomes (r) and with fibriis(f). x 110,000. Fic. 9. In this field, the keratohyalin-like material is surronnded by ribosomes (r) but is not in con- tact with any fibrils in the plane of section. x 100.000. FtG. 10. Part of the nucleus (n) and cytoplasm of a a.al in the stratum granulosum is shown here. A dense keratohyalin-like body lies within the nucieoplasm. x 68,000. The intermediate cells, instead of forming a single layer, were several cells deep between the basal and superficial cells, but they contained the usual cytoplasmic organeties; However, their complement of ribosomes was greater the nearer they were to the free surface of the epithelium. The, intermediate cells immediately below the superficial granule-containing layer contained, in addition, a few large heterogeneous bodies and many membrane-bounded vacuoles (Fig. 14). Fte. 11. A large keratohyatin-like mass in the stratum granulosum of cornified bladder is shown in this field. There are fibrils (f) in the adjacent cytoplasm, and ribosomes (r) are in contact with one edge of the dense mass. The keratohyalin-like material is composed of an irregularly dense, finely granular substance, in which no fibril,'matrix pattern can be detected. -• 130,000. Fto. 12. Part of the stratum corneum of cornified bladder epithelium. The flattened cells are bound- ed by thick, dense membranes, on which modified desmosomes (d) may be seen. A dense granular material is concentrated in the extracellular space in the region of the desmosomes and extends, in a more attenuated form, into the rest of the extracellular space. The cytoplasm of the cornified cells is filled with dense filaments. Two irregularly shaped dense areas, which may represent degenerating cell organelles, can be seen to the lower left of the field (arrows). x 53,000. M ~. 1C ~r ~ .?_.~'~.I Ls,~.• -` . ~" ~ ~~ S' ' ' y ~ i ~ _a ' ,:~a iti - - - ..'rr -.44 a..~ ~. +. ' ~ ' ,~ ~ ~ 4 '' 0 I
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© ~az .1;~ 218 R. M. HiCKS Some superficial cells showed little change from those in normal bladders wliile others were partially desquamated and contained irregular dense masses of material. The cells showing the least change (Figs. 14, 17, and 18), like those in normal transi- tional epithelium were bounded by an asymmetric membrane on their luniinal face. However, they_contained more fusiform vacuoles than are usually seen in the super- ficial cells of normal bladder (13. 15), and also great numbers of ribosomes. The latter were either clustered together in groups (Fig. 17) or filled quite large areas of the cytoplasm (Fig. 18). Between the vacuoles and ribosomes were many randomly dis- tributed tonofilaments, but very few large heterogeneous bodies were found in these cells. Most superficial cells, however, showed gross changes, and even at a relatively low magnification dark irregularly shaped osmiophilic bodies, about 0.2-0.51e in diameter could be seen in their cytoplasm (Figs. 14 and 15). These bodies correspond to the previously described granules which have the same staining characteristics as kerato- hyalin (16). Figs. 15 and 16 show the close correspondence between the images seen in the light and electron microscopes when sections of the same material were stained with lead and uranyl salts or toluidine blue, respectively. In many cells the dark bodies coalesced at the leading surface of the cell and"were confluent with the edge of the desquamated mass (Fig. 14). The desquamated mass itself contained recogniz- able cell debris including randomly oriented fibrillar material, distorted mitochon- dria, nuclear fragments, and many membranes, mainly in the form of fusiform vacu- oles. The whole mass was infiltrated with a dark homogeneous matrix with which the dark bodies were continuous (Fig. 14). The detailed morphology of the dark bodies is shown in Fig. 19. They appeared to be formed from aggregates of ribonucleoprotein particles, which in some cells were loosely clustered together (Figs. 17 and 18) and in others more tightly packed (Fig. 19). The particles were strongly stained by uranyl acetate alone as well as by lead salts; which indicates that they were not glycogen. The clusters were more electron opaque the nearer they lay to the free cell surface (Fig. 19). In some clusters the profiles of individual ribosomes appeared blurred; in others, the granular structure of the dark body was completely lost and it appeared as a homogeneous, dense mass (Fig. 19). The most homogeneous dark bodies were very similar in appearance to some of those found in cornified areas of bladder epithe- Iium (Fig. 9), but in general their substructure remained more coarsely granular than that of the fibril-associated, keratohyalin-like material of the cornified areas. The dark bodies were never observed to be limited by a membrane, and no direct association Fto. 13. Part of the basal epithelium of a noncornified area of vitamin A-deficient bladder. The basal epithelial cells (8) are separated from the underlying blood capillaries (C) and fibroblasts (F) by a thin basement membrane (b). The capillary endothelial cells (E) are relatively thick. Two bundle cells (Z) lie in the intermediate spaces between the epithelial cells. < 9000. .-, -1 0 . si 4 •w z ~ i I . F. . OS 10 r !V V
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---
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between the dark bodies and filaments was detected, although many filaments lay around and between them. Most filaments in these cells had an apparently, random orientation in the cytoplasm, but occasionally small fibrils composed of aggregated filaments were observed (Fig. 18). The nuclei of those cells producing large numbers of ribosomes had an unusual appearance (Fig. 15). There was margination and clumping of the chromatin and an overall reduction of electron opacity in the nucleoplasm between the clumps. A fur- ther unusual feature was the presence of dense granular material in the nucleoplasm. As in the normal bladder, centrioles could be found in the dells at all levels, and persisted even in the dying cells at the luminal edge of the epithelium (Fig. 19). DISCUSSION "The term 'keratinization' has unfortunately acquired somewhat different mean- ings according to the viewpoint and interests of the observer. Where definitions are not exact, confusions and misunderstandings are bound to arise. One unforgiveable and oft committed sin, is to refer to the horny layer as keratin. The terms are not equatable" (17). This , quotation from Kligman's article in "The Epidermis" clearly indicates the necessity of defi.ning terms before considering the keratinization of any tissue. The definition used in this discussion is based on that proposed by Mercer, Munger, Rogers and Roth (22). They define keratins as "proteins produced by epithelial cells, and usually retained within the cell. They are insoluble in the usual protein solvents, due to the presence of numerous disulphide bonds between peptide chains. Keratins may be predominantly filamentous, predominantly amorphous or mixtures of filamentous and amorphous elements. This characterisation is broad and does not limit keratin to one specific protein. It does not place any limits on the type of molecular structure present in keratins: ' The term "keratinization" is then taken to mean "literally the synthesis of a peculiar protein and not the synonym for horny layer formation" (17). For this study, cornification of the bladder was induced by maintaining animals on a vitamin A-deficient diet, but it could equally well have been produced by, implan- Fia. 14. Field showing the junction of an area of granular epithelium with desquamating cell debris. An intermediate cell (1) at the left is separated from the bladder lumen (L) by a granule-containing squamous cell (S). Another squamous cell (S') at the top left of the field shows little sign of dark body formation but at higher magnification could be seen to contain an unusually large number of ribo- somes. The two modified squamous cells (S). in the center and top right of the tield contain many dark bodies (k) which appear to coalesce at the free celll surface to form dense, homogeneous masses. In two places (arrows) the dense mass in the squamous cell is continuous with the dense matrix (A-f) of the desquamated material in the bladder lumen. This desquamated material also contains the . remains of a bundle cell (Z) and other cellular debris including fusiform vacuoles and filaments, which are embedded in the matrix. x 9000. it
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tation of a foreign body in the bladder with or without administration of tstradiol plus hexestrol (1). (The mode of action of vitamin A on the bladder epithelium is not directly relevant to this discussion and will be considered elsewhere.) The variation in response by the bladder epithelium to vitamin A deficiency has already been described at the light microscope level (16) and is characterized by the appearance of keratohyalin-like granules plus or minus a stratum corneum of bire- fringent fibrils. Examination in the electron microscope of patches of cornified bladder epithelium showed these areas to have a very comparable fine structure to that of mammalian epidermis. Thus the cells of the stratum basale contain numerous fibrils composed of bundles of fine filaments. A stratum spinosum is well developed in these animals, but never seen in normal bladder epithelium; and in this layer the charac- teristic cytoplasmic fibrils are found which lead into desmosomes on the membranes of adjacent cell processes. As in skin these fibrils are birefringent in polarized light (16). The laminated membrane-coating bodies and occasional tight junction between prickle cell processes (Fig. 7) are also characteristic of epidermis. The function of the tight junctions in this situation is not obvious, but in other tissues they have been shown to be the regions of maximum permeability between cells. The stratum granulosum, which was shown in the light microscope to contain material with the staining properties of keratoh}alin (16), in the electron microscope is seen to contain areas of dense amorphous material. This material is sometimes intimately associated with the filaments which compose the fibrils (Figs. 5 and 8), and it then resembles skin keratohyalin. Alternatively it may lie free in the cytoplasm (Fig. 9) with little or no association with cytoplasmic fibrils at least in the plane of section. and it then more closely resembles the keratohyalin of rat oral mucosa as illustrated by others (32). Keratohyalin-like material is also found in the nuclei of some of the cells in the stratum granulosum, and in this situation it is apparent that it is not contiguous with cytoplasmic filaments. The keratohyalin-like material in the cytoplasm is regularly associated with layers of ribosomes, irrespective of any connection with fibrils. The stratum corneum is more loosely packed than in epidermis, but is birefringent in polarized light (16) and characteristically contains filaments, no nuclei, and dense material in the intercellular spaces particularly in the region of the desmosomes. Etc. 15. A thin section through a group of intermediate cells (It-1,) and a modified squamous cell (S). The squamous cell has dense basal cytoplasm packed with ribosomes, while the distal cytoplasm contains many dark bodies (k). The nucleus of this cell has an unusual appearance with peripheral and clumped chromatin masses. Between the chromatin the nucleoplasm is pale, apart from clusters of very dense, small granules. 1 12,500. FtG. 16. A 2Ea-thick section from the same series but not immediately adjacent to that shown in Fig. 15. The same group of 4 intermediate cells (ft-1,) and a squamous cel! (S) are shown. The section was stained with toluidine blue, and the cytoplasm of the squamous cell appears strongly basophilic. x 2500. .. S.
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In the noncornified areas of granular hyperplasia, which may be found adjacent to cornified patches in the same bladder, the organization of the tissue is different and the intermediate cells do not develop the marked desmosome%fibril association seen in the prickle cells of the cornified areas and of skin, although they always con- tain many randomly oriented filaments. These areas are believed to represent an earlier stase in the differentiation of the epithelium toward cornification, for they retain more of the characteristic features of normal transitional epithelium, such as bundle cells and fusiform vacuoles, than do the cornified areas. Unlike the normal epithelium, however, they have a well developed stratum sranulosum which also contains a material with the staining properties of keratohyalin (16). In the electron microscope it can clearly be seen that the dense amorphous material in these areas is derived from aggregates of ribosomes, which become progressively more condensed until their morphology changes from a granular to an amorphous form which looks similar to, but not identical with, most of the keratohyalin-like material in the corni- pied areas. There is no association of these dark bodies with organized fibrils such as is seen in the cornified areas, but nevertheless it also may be regarded as keratohyalin- like material because of its similar staining properties (16). The noncornified areas of granular hyperplasia thus differ primarily from the cor- nified areas by a lack of organized fibrils, for there is no evidence to suggest that the free-laying dark bodies differ chemically from those which are fibril associate3. The chemical reactions underlying the change in ribosome morphology from a granular to an amorphous form have not been investigated, but it is possible that there is an enzymatic degradation of the ribonucleic acid similar to that reported for leaf ribo- somes (29). The finding of a metal, probably calcium andfor magttesium, in both keratohyalin and the bladder eranules (16) is consistent with this vie%y, for ribosomes are known to contain Mg=~ ions, as well as various basic proteins and ribonucleic acid. Nucleoprotein isolated from leaf microsomes contains ribonuclease, calcium, and magnesium in association with the ribonucleoprotein (30). Ribonuclease activity has also been observed in the lower layers of the stratum corneum of human skin (35, 37), and further enzymatic degradation of keratohyalin into its simpler consti- tuents would result in a loss of electron opacity. Just such a decrease in density is ob- served in the cornified areas by comparison of the keratohyalin in the stratum granu- losum with the overall density of the stratum corneum. It has been suggested by Bern- Ftc. 17. Cytoplasm of a superficial squamous cell from a noncornified area of the bladder. Since this tissu. was contrast stained with uranyl acetate only. which does not normally stain glycogen, the clumps of granules (r) are thought to be ribosomes. Also present in this field are fusiform vacuo- les (v) and randomly arranged filaments (r). 45,000. Ftc. 18. Part of another superficial squamous cell from the same area. The cytoplasm is packed with large numbers of ribosomes (r) and also contains bundles of filaments (t) and fusiform vacuoles (v). This tissue was contrast stained with both uranyt and lead salts. f 45,000. (,l
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stein (3) that, in skin, ribonucleic acid is degraded immediately after arrival in the stratum corneum, thus explaining why it cannot be detected in this layer. In the non- cornified areas of the bladder this further breakdown is not apparent, and the homo- geneous mass derived from the ribosomes forms a dense matrix around other des- quamated cell debris in the bladder lumen (Fig. 14). It is noteworthy that a close association of ribosomes with keratohyalin has also been reported for skin and oral mucous membrane (6, 25, 31, 32). Montagna (IS) states that keratohyalin granules in skin are surrounded by a film of nucleoprotein, and that, it is this film which is removed by treatment with ribonuclease. Brody (4) initially described keratohyalin in guinea pig as having a fine, granular substructure, so anticipating the results reported here, but tater (6, 7) regarded it as strongly staining areas of fibrils. He suggests, however, that the very opaque interfilamentous material in the keratohyalin region of the fibrils in skin might be derived from small opaque particles which, "appear identical to the ribosome particles in size and staining pro- perties" and which have "about the same high opacity as the keratohyalin regions of the tonofibrils" (6). The findings reported here do not conflict w ith this interpretation. Others have maintained that keratohyalin is a degeneration by-product of keratiniz- ing cells (1©. 36). The derivation of skin keratohyalin has thus been the subject of much spzculation, but consideration of the observations reported here together with the earlier cytochemical staining results (16), suggests that in the bladder the kerato- hyalin-like material in noncornified areas is largely derived from condensates of mor- phologically altered ribosomes. The keratohyalin-like material in the cornified areas, which closely resembles skin keratohyalin, is also surrounded by aggregated ribo- somes, and by analogy it seems possible that in this situation also, it is derived from degenerating ribosomes. The keratohyalin-like masses within the nucleus may con- ceivably be derived from nuclear riboaucleic acid-protein complexes. The clumping of chromatin and appearance of granules in; the nucleoplasm of peripheral cells in the noncornified areas of epithelium,; probably reflect metabolic disturbances associated with the onset of cell death, for such changes also occur in transitionaCepitheliat cells-rendered necrotic by cyclophosphatttide (18). Similar nu- clear changes occur in many cell types and appear to be independent of the cause of death, for they have been observed in virus-infected and irradiated cells and in many tumors. Fto.19. The luminal edge of a superficial cell in vitamin A-deficient, noncornified bladder epithelium. Clusters of free ribosomes (r) are.shown at the left of the field. In tk~.centerft the clusters still contain individual particles, but these are now more closely aggregated and very electronopaque. At the luminal edge of the cell are dark bodies (k) formed of electronopaque material which no longer has a distinct, particulate morphology. The limiting membrane of this cell has disintegrated, and the' more superficial dark bodies are in direct contact with the_bladder lumen tL). In other fields (Fig. 14), the dark bodies aie con8uent with, and appear to be the origin of, the dense, amorphous matrix of the desquamated debris: At the lower right of the field is a centriok (c,}. , 60,000 . I
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228 R M. HICKS The two main features of the normal keratinization process in skin are, first, the synthesis of x-keratin filaments and their aggregation into birefringent fibrils, and. second, the formation of keratohyalin. In certain circumstances, e.g., psoriatic para- keratosis, the first may proceed independently of the second (5). In the noncornified areas of vitamin A-deficient bladders, the second proceeds independently of the first. This suggests that formation of fibrils and formation of keratohyalin are two mutaully independent but normally concurrent processes in keratinizing stratified squamous epithelia. Where they are concurrent, the keratohyalin may infiltrate, or permeate between the fibrils, but if produced in the absence of fibrils, it forms discrete granules as shown here. These observations lend further support to an earlier conclusion. namely that the transitional epithelium lining the rat urinary bladder may properly be regarded as a keratinizing epithelium in which cornification does not normally occur (14). In the normal bladder this epithelium presents a permeability barrier to water transfer which is passive and independent of oxygen or nutrients supplied to the tissue (14). Its cells are packed with fine ca. 80 A diameter filaments (13, 1-1) which are morphologically indistinguishable from the cytoplasmic filaments in the basal cells of skin (6), kera- tiniuing tissues such as the esophageal and oral mucosae (23, 31, 32), and other non- cornifying epithelial cells such as thyroid and endothelial cells. These cytoplasmic filaments are believed by many authors to be the direct'precursors of the keratin fibrils in the epidermis, esophageal and oral mucosae (6, 28, 31), and are frequently referred to as x-keratin in these tissues. As shown here they also appear to be the precursors of the fibrils in cornified bladder epithelium. There is a concentration of disulfide groups at the luminal edge of the transitional epithelium in normal bladder (14), where the cytoplasmic filaments are most numerous and the cells are limited by an asymmetrically thickened membrane (13, 14). Treatment with thioglycolate, which splits disulfide links, ruptures this asymmetric membrane and also destroys the passive permeability barrier function of the epithelium (14). On the basis of these observations, and the fact that keratin unlike other disulfide-containing proteins is incorporated in all those vertebrate cuticles that are known to be relatively imper- meable to water (21), it was proposed that the impermeability of this epithelium might be due in part to molecular keratin, both in the form of cytoplasmic x-keratin fila- ments and possibly also incorporated as the protein moiety in the lipoprotein struc- ture of the thick surface membrane (13, 14). The bladder epithelium, like that of the oral, esophageat, and vaginal mucosae, may also be induced by mechanical stress to produce a stratum corncum. Thus, the esophagus, which is noncornified in man, is normally cornified in those animals, such as rat, pig, cow, sheep, and horse, which eat predominantly hard diets, and cornifi- cation of the bladder, leukoplakia, is observed clinically to be associated with irritz-
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ljtst, the , and para- rnified Jtt first. utaully u$mous rmeate gtanules that the ed as a o In the r which A"Its cells ogically kera- her non- plasmic the keratin frequently to be the ration of bladder limited ycolate, destroys of thesc proteins is ~ely imper- itim miaht tratin &la- otein struc- I mucosae, Ttus, the imals, such utd cornifi- !tvith irrit2- s... s. t t FINE STRUCTURE OF VITAMIN A-DEFICIENT BLADDER 4, 229 tion by urinary calculi. Furthermore, like the bladder epithelium, these other muco- sae may be induced to form a stratum corneum in animals maintained on a vitamin A-deficient diet (26, 38, 39). If the bladder epithelium, like that of the human mouth, esophagus, and'vagina, is indeed a keratin-synthesizing tissue that does not normally cornify, then the devel- opment of a stratum corneum may be regarded as a nonspecific response to stress involving hyperactivity of existing keratin-synthesizing mechanisms, rather than true metaplasia. This investigation was supported by the British Empire Cancer Campaign for Research. I wish to thank Dr. M. A. Epstein for his advice and encouragement, and Mr. G. R. Ball and Mr. T. W. Heather for their invaluable technical assistance. REFERENCES 1. ANGRIST, A., CAPL=RRO, P. and MoU%tc,s, B., Cancer Res. 20, 568 (1960). 2. BERN, H. A., Nature 174, 509 (1954). 3. BERNSTEt•t, I. A., in MONTAGNA, W. and LoBiTZ, W. C. (Eds.), The Epidermis, p. 471. Academic Press, New York, 1964. 4. BRODY, L, J. Ulrrastruct. Res. 3. 84 (1959). 5. - ibid. 6, 341 (1962). 6. --- in MoNTAGxA, W. and LostTz, W. C. (Eds.), The Epidermis, p. 251. Academic Press, New York, 1964. 7. - ibid p. 551. 8. CAPURRO, P., ANGRIST, A., BLACK, J. and ?~IOU144GIS, B., Cancer Res. 20, 563 (1960). 9. FARBMAN, A. L, J. Cell Biol. 21, 491 (1964). 10. FLESCH, P., J. Soe. Cosmetic Chemists 7, 521 (1956). 11. FREt, J. V. and SHELDO.t, H., J. Biophys. Biochem. Cytol. 11, 719 (1961). 12. FRtTHIOf, L. and WERSALL, J., J. Ultrastruct. Res. 12, 371 (1965). 13. HtcKS, R. M., J. Cell Biot. 26, 25 (1965). 14. - ibid. 28, 21 (1966). 15. ---- ibld 30,623 (1966). 16. - unpublished data. 17. KLIGMAN, A. M., in Mo~z'AGNA, W. and Loerrz, W. C. (Eds.), The Epidermis, p. 387. Academic Press, New York, 1964. 18. Koss, L. G., Lab. lnvest. 16, 44 (1967). 19. MATOLTSY, A. G., in FLORKIN, M. and tiLAsoN, H. S. (Eds.), Comparative Biochemistry, p. 343. Academic Press. New York, 1962. 20. - J. Ultrastruct. Res. 15, 510 (1966). 21. MERCER, E. H., Modem Trends in Physiological Sciences, No. 12, p. 43. Pergamon, 22. 23. London, 1961. MERCER, E. H., MUNGER. B. L, RoGERs, G. E: and RorH, S. L, Nature 201, 367 (1964). MEYER, J. and MEDAK, H., in BUTCHER, E. 0. and SOGNNAES, R. F. (Eds.), Fundamen- tals of Keratinization, Publ. 70, p. 139. Am. Assoc. Advance. Sci., Washington, ~ D.Cy 1962. :
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. . . 230 . • R. M. HICKS 24. MiLLON1G, G., J. Appl. Phys. 32, 1637 (1961). 25. MorrraGNA, W., The Structure and Function of Skin, 2nd Ed., p. 63. Academic Press, New York, 1962. 26. MoRt, S., Bull. John Hopkins Hosp. 33, 357 (1922). 27. ODLA,`+D, G. F., J. Invest. Dermatol. 34, 11 (1960). 28. - in MONTAGNA, W. and Loatrz, W. C. (Eds.), The Epidermis, p. 237. Academic Press, New York, 1964. 29. PuuE, N. W., Biochem. J. 47, 614 (1950). 30. - Biokhimiya 22, 140 (1957). 31. RHODIN, J. A. G. and REtTH, E. J., in BLTCHER, E. D. and SoctiNAES, R. F. (Eds.), Fundamentals of Keratinization, Pubt. 70, p. 61. Am. Assoc. Advance. Sci. Wash- ington, D.C., 1962. 32. ROGERs, G. E., in MONTAGNA, W. and Loattz, W. C. (Eds.), The Epidermis, p. 179. Academic Press, New York, 1964. 33. RoTH, S. 1. and CLARK, W. H., in MOrTaGNA, W. and LostTz, W. C. (Eds.), The Epi- dermis, p. 303. Academic Press, New York, 1964. 34. S.asATtNt, D. D., BENSCH, K. G. and SARR-,Err, R. J., J. Cell Biol. 17,19 (1963). 35. SANTOtatit, P. and RorxM,aN, S., J. Invest. DermatoL 37,489 (1961). 36. SELBY, C. C., J. Biophys. Biochem. Cytol. 1, 429 (1955): 37. STEIGLEDER, G. K. and RAAB, W. P.. J. Invest. Dermatot. 38, 209 (1962). 38. TYsov, M. D. and SHrrH. A. H.. Am. J. PathoL 5, 57 (1929). 39. WOLBACH, S. B., and HowE, P. R., J. EYptt. Med. 42, 753 (1925). Ift
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