MACRONUCLEAR DIVISIONI N Blepharisma 67. ___ 1971. Spatial discrimination in the cytoplasm during microtubule morphogenesis. Nature ( L o n d o n ) 232, 38789. 68. ____ 1972. Microtubule-arms and propulsion of food particles inside a large feeding organelle in the ciliate Phascolodon vorticella. I . Cell Sci. 10, 883-903. 69. Wilfert M. 1972. Zytologische Untersuchungen an dem Ciliaten Blepharisma americanurn Suzuki 1954, Stamm Berlin (Heterotrichica. SDirostomatidae) sowie Bemerkungen zur Taxonomie und Systema’tik der Gattung Blepharisma PeGy 1849. Arch. Protistenk. 114, 152-230. 70. Wilson L. 1970. Properties of colchicine binding protein from chick embryo brain: Interactions with vinca aliaioids and podophyllotoxin. Biochemistry 9, 4999-5007. 71. ~- 1975. Microtubules as drug receptors: Pharmacological properties of microtubule protein. A n n . N . Y . A c a d . Sci. 253, 213-31. 72. , Bamburg JR, Mizel SB, Grisham LM, Creswell KM. 1974. Interaction of drugs with microtubule proteins. Fed. Proc. 33, 158-66. ~
73. --, Bryan J. 1974. Biochemical and pharmacological properties of microtubules, in DuPraw EJ, ed., Advances in Cell and Molecular Biology, Academic Press, New York, pp. 21-72. 74. , Friedkin M. 1966. Biochemical events of mitosis. I. Synthesis and properties of colchicine labeled with tritium in its acetyl moiety. Biochemistry 5 , 2463-8. 75. Wunderlich F, Heumann H. 1974. I n uiuo reassembly of microtubules in the presence of intracellular colchicine. C y t o biologie 10, 140-51. 76. --, Muller R, Speth V. 1973. Direct evidence for a colchicine-induced impairment in the mobility of membrane components. Science 182, 11 36-8. 77. , Peyk D. 1969. Antimitotic agents and macronuclear division of ciliates. Naturwissenschaften 56, 285-6. 78. ---, Speth V. 1970. Antimitotic agents and macronuclear division of ciliates. IV. Reassembly of microtubules in macronuclei of Tetrahymena adapting to colchicine. Protoplarnza 70, 139-52. ~
J. I’ROTOZOOL.2 4 ( 2 ) , 275-283 (1977)
Effects of Dimethyl Sulfoxide on Tetrahymena pyriformis GL. Fine Structural
Changes and Their Reversibility* JYTTE R. NILSSON Iiistitute of General Zoology, Univerrity of Copenhagen, (iiiivei~itetsparken15, DK-2100 Copenhagen 0, Denmark
SYNOPSIS. Fine-structural changes are induced in Tetrahymena by exposure to 7.5% dimethyl sulfoxide (DMSO) in the presence of growth medium. Some of these changes (nucleolar, mitochondrial, peroxisomal) resemble those seen during starvation, in agreement with the previously reported inhibitory effect of DMSO on food-vacuole formation ; however, changes such as helical formations of polyribosomes indicate additional internal actions of the reagent. The effects vary to some extent within the same group of cells, suggesting that sensitivity to the reagent may differ with the stage in the cell cycle. The structural changes induced by a 1-hr exposure to DMSO are reversible, but recovery of the cells after removal of the reagent is slower than that seen after starvation. The observations suggest that the recovery is associated with renewed synthesis.
Index Key Words : Tetrahymena pyriformis; dimethyl sulfoxide (DMSO) ; reversibility of fine-structural changes.
IMETHYL sulfoxide (DMSO) is a dipolar aprotic solvent with wide biologic applications (20, 21, 25). The reagent is used in cryo- and radiobiology ( 1, 2, 23 ) , in clinical therapy by itself, or as a drug carrier (19, 24, 48, 6 2 ) , or as a solvent of water insoluble compounds, e.g. cytochalasin B ( 5 ) . DMSO has properties resembling those of water with which it is fully miscible. I t readily accepts protons (46, 55, 56), and the hydrogen bonds formed between DMSO and water are stronger than those existing between water molecules (6, 55, 56). The biologic action of DMSO has been ascribed to an interference with the water shell around macromolecules by substituting for water, which may result in conformational changes and subsequent interference with the proper functioning of the molecules (23, 44, 46, 55, 56). Such conformational changes have been demonstrated in proteins (17, 23, 44-46), in lipids ( 3 2 ) , and in nucleic acids (45, 5 5 ) ; these changes are readily reversible after removal of the compound. The fact, however, that DMSO may be metabolized (13, 61) indicates that the physicochemical properties of this compound alone may not be responsible for its biologic action. Thus it is of special interest to study the effects of DMSO on in viva systems. Recently, DMSO has been shown to interfere with at least
-* This investigation was supported
by grants from the Carlsberg Foundation and Danish Natural Science Research Council.
3 cellular functions in T e t r a h y m e n a . It has a dose-dependent effect on food-vacuole formation, with completr inhibition in 7.5% (v/v) DMSO, a concentration within the range used in cryobiology; it disturbs the function of the contractile vacuole; and it inhibits nuclear elongation during apparently unaffected cytokinesis ( 3 7 ) . T h e present study is an extention of this lightmicroscopic work. I t was undertaken to gain morr information on the sites of action of DMSO in T f t r a h y m f n a . MATERIALS AND METHODS T e t r a h y m e n a pyrzforrnis G L was grown axenically at 28 C in 2% (w/v) proteose-peptone medium eniiched with 0.1% (w/v) liver extract and salts ( 4 3 ) . The cell density of the cultures was ascertained either microscopically in a calibrated cell chamber or automatically in a cell counter, Coultrr Counter model ZB. Cultures in the exponential growth phase (5-8 x lo4 cells/ml) were exposed to 7.5% (v/v) DMSO for 1 hr. After the exposure, DMSO was removed, the cells were washed 3 times, by centrifugation, in fresh growth medium; the cell3 were left in this medium. The experimental temperature wab 28 C. Cell samples were removed a t 10, 15, and 60 min during the 1-hr exposure to DMSO and at 10, 15, 60, 90, and 180 niin after the removal of DMSO. The cclls were examined also up to 20 hr after the exposure.
EFFECTSO F DMSO
2 76 I
90 MINUTES AFTER REMOVAL OF DMSO
TIME IN HOURS
Fig. 2. Recovery of food-vacuole forming capacity of T e t r a hynienn aftrr a 1-hr exposure to 7.570 DMSO. Results expressed as the number of vacuoles formed during a 10-min exposure to carmine particles. Growth medium. Test of the 3 populations studied in the efectron microscope.
Fig. 1. Effects of 7.5% ( v / L ) DMSO on exponentially multiplying Tetrahymeitu. Arrow indicates time of addition of DMSO to experimental sample ( x ) and of equal volunic of water to control sample ( 0 1 , Double counts of cell density in 1 experiment.
are 10, 6, 3, 3, and 2% aftrr 1, 2, 3, 4, and 5 hr, respectively (mean values from 5 c.xpcrinients) . Observations of the cells in z.iz'o reveal that throughout the experimental period cell divisions are completed, i.e. cell separation occurs, but as preCells for electron iriic.roscopy \vt=rt- fixrcl at room tcmperature viously reportcd (37 ) , thc anterior daughter cell usually becomes in 4% (v.'v: glutaraldehyde in 0.1 h1 cacodylate buffer at pH imniobile and dies immediately upon separation. During the 7.1 (cell suspension: fixative = 1 : I ) for 10 niin. After a brief first %-hr cxposure normal nuclear elongation is observed in wash in the buffer, the cells were postfixed in 170 (w/'v) OSO, dividing cells and both daughter cells survive division; however, in thr same buffer for 1 hr. .4fter dehydration in a graded series as reportrd prrviomly ( 3 7 ) , after a 1-hr exposure 90% of of ethanols and finally in propylene oxidc, thc cells were em- the cell divisions orcur without nuclear elongation, resulting i n bedded in Epon ( 2 9 ) . T h e sectioned material Lvas stained for anuclcatc antrrior daughters (short survival) and in posterior 20 min in 7inc-uranyl acetate (60; and for 3 min in lead citrate daughter cells containing the entire macronuclrus. T h e data ( 5 8 ) . The sections \\err c.xaniined i n ;I Zeiss EM!, electron sho\vn in Fig. 1 thus conceal the fact that both cell multiplicamicroscope. tion and cell death occur. By the end of a 5-hr exposure, most When samples \vex removed for electron microscopy the ciliates are spherical in shape, with grratly enlarged contractile cells were siniultanrously tested for thrir capacity to form vacuoles, and in 80% of the aforementioned experiments all food vacuoles. The number of food vacuoles formed during a cells had died after a 24-hr rxposure. 10-min exposure to carmine particles \\as counted in 100 cells, As prrviously r t p r t e d ( 3 7 ) , no food vacuoles are formed as described prcviously (37 1. during a 10-min exposure to 7.5% DMSO. T h e rate of foodThe results were obtained from 3 inclepenclcnt rxpcrimt=nts vacuole formation returns to the control value 75-90 min after performed under identical conditions. removal of DMSO (Fig. 2 ) . Detailed analysis of the cells exposed to carmine particles reveals that this gradual recovery RESV1,TS i\ not duc to a slowly increasing rate of vacuole formation in Ligh/-Microscoflic Obscrr!ntions.-l'c,ti-cihy,nt,na has little tol- individual cells but to a gradual increase in the percentage of erance to prolonged exposure to 7.5% DMSO. few cells surviv- cells forming vacuoles. Thus 15 and 30 min after removal of DMSO only 20% and GO%, respectively, of the cells form food ing a 21-hr exposure (37). Furthrrmore, the cell density of DMSO-treated cultures dors not increase much, even though \acuoles. I n contrast, 15 niin after transfer to fresh medium the Percentage of dividing cells after a 1-hr exposure is the same of 1-3-hr stanzed cells all nondividing cells form food vacuoles. The percentage of dividing cells after a 1-hr exposure to 7.5% as that of control cells ( 3 7 ) . The effect of DMSO on the cell density of exponentially multiplying cultures was therefore DMSO corresponds to that found in the control sample, i.e. 10%. After removal of DMSO few dividing cells are observrd studied during a 5-hr exposure to 7.570 DMSO; the results of 1 experiment are sho\vn in Fig. 1. Little increase in the cell during the first 2 hr, but an hour later cell divisions occur at density is evident, VLWI though the percentages of dividing cells a slightly increased rate ( - 15%). Whereas nuclear elongation
+ Fig. 3. Control cell (interphase) from the logarithmic growth phase. The macronucleus ( n u ) contains numerous small nucleoli ( n ) . dispersed chromatin granules (arrows), and nucleoplasm of low electron density. Food vacuole. fv ; mitochondrion, m ; peroxisonie, p ; nuclear envelope. ne : and rough endoplasniic reticulum, er. x 27,000. Figs. 4. 5 . [Cells after 1 hr in 7.5% DMSO. x 27.000.1 4. Aggregation of nucleoli ( n ) and detachment of the granular nucleolar
material (arrows) are evident. Note also the condensed chromatin granules (ch) and increase in density of the nucleoplasm over that in control cells (cf. Fig. 3 ) . The rough endoplasmic reticulum (er) and the perinuclear space (ne) appear swollen. Lipid droplet, 1; small vacuole, av; peroxisome, p ; mitochondrion, m. 5. Nucleolar changes are more pronounced than in Fig. 4. Much granular material (arrows) has become detached from the large nucleolar fusion bodies ( n ) . Note the condensed chromatin granules (ch) in electron-dense nucleoplasm, the nuclear envelope (ne) with enlarged perinuclear space, and the small vacuoles ( a v ) .
Fig. 6. Control cell from logarithmic growth phase. Typical structure of mitochondria ( m ) and peroxisomes ( p ) is evident. Both organellrs are of the “loose“ type. i.e. they are of low electron density and hase much matrix space. Rough endoplasmic reticulum, er. x 60.000. Figs. 7. 8. [Cells after 1 hr in 7 . 5 % DMSO.] 7. Mitochondria ( m ) and peroxisomes ( p ) are of the “dense” type, i.e. they have elertron-dense matrix. Enlarged cisternae of the endoplasmic reticulum, er. x 60,000. 8. Several helical arrays of ribosomes (arrows) which may represent polyribosomes are evident. T h e nucleoli ( n ) are slightly aggregated. Note the nuclear envelope ( n e ) with enlarged perinurlear space. x 70,000.
is inhibited in dividing cclls in I ~ h f S O only , 5% of the dividing cells undergo abnormal nuclear division 3 hr after removal of DMSO. Further, feiver than 1% dead (or abnormal) cells are srcn 5 hr after removal of the cornpound; after 20 hr the proliferating population appears normal. Elrctron-Microscopic 0hsercations.-The fine structure of the control cells is typical of Tetrahyvic~rtafrom cxponentially multiplying cultures. The crlls contain food \.acuoles, and there are numerous small (0.3 pi1 ) nnclcoli juxtaposed to the nuclear tm,clope f Fig. 3 ). Furthcrmorc, thc mitochondria and the peroxisomes have low electron density Fig. G ! , i.e. they are of the “loose” type ( 3 5 ) with much ntatr x space. Typically cells in interphase (the majority) have “dispersed” chromatin ( 3 4 ) , i.e. much filamentous material radiates from the chromatin granules. As demonstrated previously ( 3 6 ) . this chromatin confiquration is correlated u i t h activity in DNA andlor RNA synthesis (cf. Ref. 3 8 ) . l h r w is no indication of autophagic vacuoles, lipid droplets, or glycogen particles. C d l s exposed to i.570 DMSO for 1 hr undergo structural alterations. Chaiiscs occur in prc-existing. organelles and, in addition, new cell constituents appear; typically, no large food vacuoles are wen, and the contractile vacuole is often much twlarged. Thc nuclmlar organization is changed in all cells but to a varying cxtent e \ ~ nLvithin the same cell sample. In sonic cells f 10 of 31’1, larger nucleolar apgrepites, or fusion bodies, may hr sern (Fig. 5 ) , \vhcreas only slight aggregation of the small nucleoli is seen in other cells !Fig. 4 ) ; in all cases granular matcrial has become detachrd from the nucleoli (Figs. 4, 5 ; cf. Fig. 3 ) . The chromatin granules appear smooth in outline, i.e. they have little radiating. filamentous material, as is typical of starvrd cells ‘ 2 2 , 34, 3 8 ’ . In a fen. cells ( 3 of 311, somp niicrotubular niatc~rial ha5 brcn observed in the macroriricleus, but this was much less frrquent than that normally seen i n a dividing nucleus. The cisttrnae of the rough endoplasniic rr~ticulum,including the perinuclrar space: are enlarged in most cc.11~ (Figs. 4, 5. 8 ) . Il’hether this phenomenon is correlated ith the enlarsed contractile vacuole is unknon-ti, but no direct c o n n r d o n has becn obrcrvrd betnwn the 2 conipartnicnts. T h e mitochondria and the pcroxisonies are niore electron dense (Fig. i ) than those in the c.ontrol cells (Fig. 6 ) . tvith electron-dense matrix f 35). Mitochondria may also occasionally have atypical arrangement of their tubrili. In many of the cells (20 of 31 1 some ribosomes form distinct aggregatcs. which appear to be hdical configurations of polyribosonies (Fig. 8 ) . NeIv cell constitucmts such as lipid droplets and small vacuoles (Fig. 1) apprar. The latter structures may be autophagic in nature, although the c p p l a s n i i c origin of their contents is rarely recognizable. Most of the descrihd changes niay be observed in cells exposrd to DMSO for only 10 or 15 min. No structural alteration could be observed in tht. cytnstonial region to explain the inhibition of fooci-vacuolr formation. Special attention was paid to the “specialized cytoplasm” ‘‘4 1 surrounding the cytostorne. This rcgion, composed of a nrtbvork of 5-nIll thick (actin-like) filaments, is the same in DRISO-treated and control cells. T h e DMSOtreated individuals. however, contained numerous small vesicles n w r the cytopharyn~ealmcmbranr, as is typical of starved cclls
!35, SSj. No apparent change in the pores of the contractile vacuole could be observed to explain the interference with the rate of expulsion. After removal of DMSO, there is a gradual reversal of the structural changes induced by this compound. The recovery is accompanied by additional alterations. During the early period of recovery (15 m i n ) , the mitochondria are very electron-dense, i.e. they appear contracted, whereas the peroxisomes are even less electron-dense than those of the control cells; these extreme changes may reflect an osmotic shock caused by the removal of DhlSO. The contractile vacuole function is restored by this time ( 3 7 ) . After 30 min of recovery, the cells still contain lipid droplets, and glycogen particles are seen (Fig. 9 ) ; the mitochondria and the peroxisonies are both of the “dense” type (Fig. 9 ) . Food vacuoles are evident in some cells, whereas other cells contain autophagic vacuoles in some of which the cytoplasmic origin of the contents is identifiable; furthermore, most cells contain dense granules. A gradual reversal of the nucleolar organization to the state characteristic of the control cells is reached 1 hr after removal of DMSO. At this time the chromatin granules ha\re more radiating material than during the exposure to DMSO. Although the nuclear structures appear normal (e.g. microtubules are abundant in some nuclei in agreement \vith the increase in the number of dividing cells), the cytoplasm has not reverted to the state of the controls. \\’hereas the mitochondria are of the “loose” type, the peroxisonies are still of the “dense” type (Fig. 10). Further, lipid droplets and dense granules, as well as glycogen particles are present. T h e outline of the lipid droplets is irregular (Fig. l o ) , i.e. they are apparently in a state of resorption, and their position near peroxisomes suggests the occurrence of gluconeogenesis. The reported results are based on observations from 10 cells per sample from 3 individual experiments. I t should be stressed that, as also pointed out above, some variation is found Lvithin each saniple; evidently DMSO affects some cells more than others. This difference in sensitivity to the reagent could conceivably be related to different stages in the cell cycle; however. such a relationship has not becn established in the present study.
DISCUSSION DhiSO has bcen shown to interfere with metabolism. At low concentrations it induces cell differentiation in virus-infected lrukemic cells ( 1 1, 271, and at high concentrations it has a dosedependent effect on cell growth in general (3, 11, 12, 16, 47), morc specifically, on DNA and RNA synthesis (1, 14-16), protcin synthesis ( 1, 49) and the rate of oxygen consumption (10). This biologic action of DMSO may be responsible for its radioprotective nature ( 1) . .is shown in the present study, DMSO a t a concentration of 7.5% induces structural changes in exponentially multiplying T e t m h s m m z . Some of the induced structural changes, i.e. the altered nucleolar organization, the altered structure of mitochondria and peroxisomes, and the appearance of lipid droplets, niay also tie observed in Telrnhyinena during starvation, during the stationary growth phase, or during exposure to actinomycin
c Fig. 9. Cell 30 min after renio\al of DMSO. The nucleoli ( n ) are still fused, and the detached granular material (nl) is still present: the chromatin granules ( c h ) are condensed. The mitochondria ( m ) and peroxisornes ( p ) are of the “dense” type. Clusters of glycogen particles are indicatrd by arrows. Lipid droplets, 1; nuclear rnvrlope, ne. x 60.000. Fig. 10. Cell 3 hr after removal of DMSO. The nurleoli ( n ) have revertrd to thc structure typical of logarithmic growth phase cells ( r f . Fig. 3 ) . Thr peroxisomrs ( p ) are of thr: “dense“ type. The mitochondria, not shown in this figure, are of the “loose” type (see Fig. 6 ) . Xote thc close association between a peroxisome ( p ) and a lipid droplet ( l ) , the latter being apparently resorbed (glueoneogenesis?) (cf. with lipid droplet in Fig. 9 ) . Electron-dense granule. g. X 60,000.
EFFECTSOF DMSO D (26, 35, 38, 40, 50). These conditions are all correlated with a decreased rate of metabolism (cf. Ref. 38). The observation that the structures of DMSO-treated Tetrahymena resemble those seen in this ciliate under various conditions, indicates that the structural changes do not represent fixation artifacts in the presence of DMSO but rather reflect an altered physiologic state of the cells. Accordingly, it is evident from the present results that DMSO interferes with metabolism of Tetrahymena, as it does in other cell systems. This conclusion is supported by the reported decreased rates of oxygen consumption (54), of cell multiplication (Fig. l ) , and of RNA synthesis (39) in Tetiahymena exposed to DMSO. The induction of ribosomal aggregates (Fig. 8 ) suggests an interference with protein synthesis, as discussed below. Most of these changes could, in fact, be ascribed to starvation induced by the inhibitory effect of DMSO on food-vacuole formation. The induction of helical configurations of polyribosomes may, however, be a direct effect of DMSO because such structures have not previously been observed in Tetrahymena. Helical arrays of polyribosomes have been observed in other cells, for cxample in Amoeba proteus exposed to emetine ( 9 ) , in tissue culture cells subjected to cooling ( 7 ) , and in cysts of Entamoeba invadens (31). Emetine is an inhibitor of protein synthesis and protein synthesis is likely to be negligible in the last two examples. Flickinger ( 9 ) interprets the helical structures as inactivated polyribosomes inhibited at the level of translocation, a situation which would preserve the binding of ribosomes to mRNA. The finding of polyribosomes in this configuration suggests that DMSO affects protein synthesis in Tetrahymena, as it does in other cells. Furthermore, the formation of helical patterns of polyribosomes may be an effect of the dose, since Saborio & Koch (49) report a complete breakdown of polyribosomes in HeLa cells in 12% DMSO. That DMSO may interfere with protein synthesis in Tetrahymena has been suggested before (37). After a 1-hr exposure to DMSO, nuclcar elongation is virtually absent in dividing cells. Thus cell division results in anucleate daughter cells (short survival) and in daughter cells ( GI-cytoplasm) containing the entire macronucleus (G,-nucleus) (37). Since microtubules are involved in nuclear elongation (57), an action of DMSO on microtubular material is suggested. Furthermore, since DMSO is used as a stabilization medium for isolated microtubules ( 8 ) and since both depolymerization and repolymerization of microtubules may occur in 10% DMSO (0. Behnke, personal communication; cf. Ref. 37), the possible effect of DMSO on microtubular material was ascribed to an interference with the synthesis of microtubular subunits (37). The finding of some microtubular material in DMSO-treated cells in the present study does not contradict this assumption, since a small pool of microtubular subunits could have been present before addition of DMSO. The observation that normal nuclear elongation occurs up to about a %-hr exposure, but not after a 1-hr exposure to DMSO, suggests that microtubular material for nuclear division is synthesized between 1 hr and % hr before cell division. No structural change could be observed to explain the inhibition of food-vacuole formation; however, DMSO may interfere with the process at a submicroscopic level. The following events are essential for food-vacuole formation (38) : ( a ) incorporation of membrane into the cytopharyngeal membrane to provide the limiting membrane for a new food vacuole; and ( b ) sealing off of the filled food vacuole before it leaves the cytostome. I n both events fusion of membrane is involved. If the properties of the membrane are altered by DMSO, such membrane fusion may conceivably be prevented. The compound
may have such an effect, since it causes a redistribution of intramembranous particles (30) and interferes with the activity of some en7ymes within the membrane ( 4 ) . DMSO may also interfere with a contractile mechanism facilitating the sealing off of the food vacuole. This contraction may occur in the region around the cytostome containing “specialized cytoplasm” (38, 5-nm-thick filaments 41) which is composed of a network of (actin-like?). Such an action of DMSO is not unlikely since the compound induces a positive inotropic response in cardiac muscle probably by interfering with the availability of the calcium pool (52). Direct structural evidence for this explanation is not to be expected, because no structural change could be detected in the contractile filaments of cardiac muscle on exposure to DMSO (51). Finally, the effect of DMSO on food-vacuolc formation and on the function of the contractile vacuole, may be explained by the fact that the compound causes a decrease in the cell content of ATP (28). Typically, the effect of DMSO on biologic material is reversible after removal of this compound. The reversibility, however, is dependent on the concentration of DMSO and on the duration of exposure (17, 2 3 ) . In cryobiology, the concentrations of DMSO required for protection of the cells are usually higher than those tolerated at normal temperature (33). For example, Tetrahymena can be frozen successfully in the presence of 10% DMSO with good recovery after thawing (18, 42, 53, 59), whereas the cells have a low tolerance for this concentration at the normal qrowth temperature (37). Furthermore, cells from logarithmic-pha.;e culture5 are more sensitive to DMSO than those from the early stationary-phase cultures, i.e. tolerance for the reagent seems to depend upon a low rate of metabolism. The effects of a I-hr exposure to 7.5% DMSO are reversible in Tetrahymena. As shown in the present study, the recovery of the cells is correlated with additional structural changes, initially by the appearance of glycogen particles and of autophagic vacuoles, and later by resorption of lipid droplets (glyconeoyenesis?) (Fig.. 10). The altered structures of DMSO-treated ciliates revert to normal at a slower rate than in starved cells transfcrred to fresh growth medium. This difference is due in part to the fact that not all cells begin food-vacuole formation immediately after rpmoval of DMSO, which indicates a slow rate of removal of DMSO from the cells and/or renewed synthesis to repair damage caused by the reagent. Actually, 1 hr after removal of DMSO the rate of RNA synthesis in Tetrahymena is higher than that in cells before the exposure to DMSO ( 3 9 ) . That nutrients are required for full recovery of the DMSOtreated Tetrahymena has been indicated in a previous study (37) in which full recovery of the food-vacuole forming capacity was noted only in cells in nutrient medium but not in those in starvation medium. Of the described structural changes induced in Tetrahymena by DMSO, the formation of helical patterns of polyribosomes may be a direct effect of the compound; the other changes may represent secondary effects. Since, however, DMSO affects some functions [inhibition of food-vacuole formation, of nuclear elongation, and proper functioning of the contractile vacuole (37)] for which no structural changes could be detected, the effect of the compound may most readily be explained in terms of altered conformation of macromolecules induced by substitution of DMSO for water. This general effect may cause inactivation of some, but not all enzymes and thereby change the physiologic state of the cells to a degree conceivably dependent on cell cyclc stage. This would account for the structural changes observed, as well as for the varying response of individual cells from a population.
duction of rrvthroid differentiation bv dimethvlsulfoxide in cells infected with Friend virus: relationship to th; cell cycle. Proc. S a t . Actid. Sci. C‘SA 72, 28-32. 28. Loten EG. Teanrenaud B. 1974. Effects of cvtochalasin B. colchicine and v k r i s t i n e o n the metabolism of isolated fat-cells. Biocheni. J. 140. 185-92. 19, Luft J H . 1961. Improvements in epoxy resin embedding methods. J. Bioph)’.r. Biochen~.Cl’tol. 9, 409-14. R E F ER F. NC: F,S :30. McIntyre JA. Gilula NB, Karnovsky M J . 1974. CryoIirotectant-inducrcl redistribution of intramembranous particles in 1. Ashwood-Smith MJ. 1967. Radioprotcctive and cryopromouse lyniphocytes. J. Cell Biol. 60, 192-203. trctive properties of dimethyl sulfoxide in cellular systems. .4nn. 31. Morgan SM. Slayter HS, Weller DL. 1968. Isolation A’. Y . Acad. Sci. 141, 45-62. of ribosomes from cysts of Entarrioeba invadens. J . Cell B i d . 36, 2. 1975. Current concepts concerning radioprotec45-5 1. tive and cryoprotecti\-e properties of dimethyl sulfoxide in cel:-l suifoxide. P f o c . (phcnosrt X ) . after freezing and thawing from -196 C. J . ProtoSoc. Exp. B i d . M e d . 128: 648-50. :ool. 22. 2 3 3 - 7 . 16. Hellmann A . Farrrlly JG. Martin D H . 1967. Some bio43. Plesrwr P: Rasmussen L, Zeuthen E. 1964. Techniques logical properties of dimethyl sulfoxide. N u t u r e 213. 982-5. used in the study of synchronous Tetrahymena, in Zeuthen E, ed., 1 7 . Henderson T R . Hendrrson RF. 197.5. Effects of di- S),nchron), in Cell Dirsision and Growth, Interscience, New York, mcthyl sulfoxide on subunit proteins. A n n . iV. Y . Accid. Sci. 243. pp. 543-64. 38-53, 44. Rammler D H . 1967. T h e effect of D M S O on several 18. Hwaiig S. Da\-is EE. Alexander M T . 1964. Freezing and enzyme systems. A n n . h’. 1.. Acad. Sci. 141, 291-9. viability of Tetrah),r)ieiicc p),rifoi mi\ in dimethylsulfoxide. Science 43. 1971. Use of D M S O in enzyme-catalyzed re144, 64-5. actions. in Jacob SW. Rosenbaum E, Wood D C , eds., Dimethyl 19. Jacob SbV. 1971. Pharni,irology of DMSO, in J,icob Sulfo.side. Marcel Dekker, New York, pp. 189-206. SLY, Rosenbaum EE. IYood DC. eds.. Dimeth1,l Sulfoxidc. Marcel 46. -. Zaffaroni .4. 1967. Biological implications of Dekker. New York. pp. 99-1 1 2 . 2 0 . -~ -. Hcrschlrr R. eds. 1975. Biological Actions of D M S O based on a review of its chemical properties. A n n . N . Y . .4cnd. Sci. 141. 13-23. Dimethyl Sulfoside. .4nn. S . 1.. i l r n r l . Sci. 243. 1-508. 47. Roisen F.J. 1975. ‘I’he effects of dimethyl sulfoxide on 21. Roscnbauin EE. M’ood DC. rds. 1971. D i m e t h ~ i neurite development in ritro. A n n . N . Y . Acad. Sci. 243, 279-96. Sulfoxide. Marcel Drkker. New York. 22. Jeter J R . Pavlat l V . 4 , Cameron I L . 1975. Changes in 48. Rosenbaum EE. Herschler RT. lacob SW. 1965. Dimethyl sulfoxide in musculoskeletal diiordks. J . A m . M e d . Assoc. the nuclear acid proteins a n d chromatin structure in starved and 192. 309-13. refed ‘l’ettahyrnenn. E x p . Cell Re,. 93. 79-88. 23. Karow ;lM. 1969. Cryoprotectants-a new class of 49. Saborio TL. Koch G. 1973. Reversible inhibition of Drodrugs. 1. P h a r n i . Piicirjnncol. 21. 209-23. tein synthesis ig HeLa cells by dimethylsulfoxide. J. Biol. Chknt. 248. 8343-7. 24. Klignian A M 1963. Topical pharmacology and toxicology of dimethyl sulfoxide. J. A m . M e d . A n o c . 193. 796-80-1.. 50. Sntir B. Dirksen ER. 1971. Nucleolar aging in Tetra25. Lrake CD. c.d 1967. Biological actions of dimethyl sul- h),ti/ena during the cultural growth cycle. ]. Cell B i d . 48, 143-54. foxide. A n n . A‘. 1.. .4c(id. Sci. 141. 1-671. 51. Shlafer M. Karow AM. 1971. Ultrastructure-function correlative studies for cardiac cryopreservation. I. Hearts perfused 26. Lev)- M R . Elliott A l l . 1968. Biochcmical and ultrawith various concentrations of dimethyl sulfoxide ( D M S O ) . Cryostructural c h a n , w s in 7’etrnh),tnencz p),rijorniiy during starvation. 6iolog)’ 8. 280.9. J . Protozool. 15. ?OX-12. 52. . Matheny JL, Karow .4M. 1974. Cardiac ino2 7 . Le\-y J, Terada M. Rifkind RA. Marks P.4. 1975. I n ~~
EFFECTSOF DMSO tropism of DMSO: osniotic effects and interactions with calcium ion. Eur. J . Pharmacol. 28, 276-87. 53. Simon EM. Schneller MV. 1973. The preservation of ciliated protozoa at low temperature. Cryobiolog; 10, 421-6. 54. Skriver L, Nilsson JR. 1974. Oxygen uptake and foodvacuole formation in Tetrahymena. J . P7OtO%OOl. 21,462. 55. Szmant HH. 1971. Chemistry of DMSO, in Jacob SW, Rosenbaum EE, Wood DC, eds., Dimethyl Suljoxide, Marcel Dekker, New York, pp. 1-97. 56. __ 1975. Physical properties of dimethyl sulfoxide and its function in biological systems. A n n . N . Y . Acad. Sci. 243, 20-3. 57. Tamura S, Tsuruhara T, Watanabe Y. 1969. Function of nuclear microtubules in macronuclear division of Tetrahymena pyriformis. E x p . Cell K e s . 55, 351-8.
58. Venable JK, Coggeshall R. 1965. A simplified lead citrate stain for use in electron microscopy. J . Cell Biol. 25, 407-8. 59. Wang G-T, Marquardt WC. 1966. Survival of Tetrahymena fiyriformis and Paramecium aurelia following freezing. J . Protoeool. 13, 123-8. 60. Weinstein R, Abbiss T, Bullivant S. 1963. The use of double and triple uranyl salts as electron stains. J . Cell B i d . 19, 74A. 61. Wood DC. 1971. Fate and metabolism of DMSO, in Jacob SW, Rosenbaum EE, Wood DC, eds., Dimethyl Sulfoxide, Marcel Dekker, New York, pp. 133-45. , Wood J. 1975. Pharmacologic and biochemical 62. considerations of dimethyl sulfoxide. A n n . N . Y . Acad. Sci. 243, 7-19. ~
Bocquet, C., Gtnermont, J., Lamotte, M., eds. 1976. Les Probldmcs dc l‘Espdce duns le Rdgne Animal. T o m e I . Soc. Zool. France MCm. #38., 195, rue Saint-Jacques, 75005 Paris, France. 90 F. This is the first of 3 volumes on species problems in the animal kingdom. I t includes articles on ornithology, teleost fishes, Lepidoptera, Drosophilidae, mosquitoes, copepods, isopods, lamellibranchs and protozoa. The next volume will have chapters on the primates, rodents, lizards, modeles, gastropods, Orthoptera, Collembola and nematodes. The 3rd volume will approach the problems in a more synthetic way by considering the different types of criteria of species. The chapter on protozoa was written by GCnermont. I t is de-
voted primarily to Paramecium aurelia and Euplotcs spp. I t discusses Sonneborn’s (1975) 14 sibling species of P. aurclia and asks the question whether it would be progress to replace “P. aurelia species I ” (or, more simply P. aurelia-I) by “P. primaurelia,” etc. GCnermont’s answer is, “Ce n’est pas sfir.” So far as asexual reproduction is concerned, GCnermont says simply that the biologic notion of species is incompatiblr with the absence of sexual reproduction. Yet sexual reproduction is rare among flagellates and sarcodines, and Gknermont says that if it occurred among the trypanosomes it would surely have been discovered by now. I guess the conclusion i5 that there arc no species among nonsexual organisms; surely we shouldn’t discommode the ornithologists and entomologists by insisting that there are.-NORMAN D. LEVINE, Collcge of Vcterinary Mcdicine, Uniucrity of Illinois, Urbana, I L 61801, USA. .
Anderson, J. M. & Macfayden, A., eds. 1976. T h e R o l e of T c r restrial and Aquatic Organisms in Decomposition Processes. Blackwell Scientific Publishing Co., London. ix 474 pp. $32.50.
Protozoa must be considered part of the decomposer community because their predatory activities enhance bacterial degradation. Therefore, comprehension of decomposition processes is necessary to understand protozoan ecology. This book, like a number of other recent symposia on decomposition, emphasizes the functional roles of organisms in nutrient recycling processes, rather than detailed aspect4 of organism biology. T h e book differs from other similar symposia in its broad coverage of habitats: coastal surface, macrophyte, and bottom marine habitats; estuaries; lakes and streams; and a spectrum of terrestrial habitats from tundra to tropical. The 19 chapters are arranged into 4 Sections: physico-chemical aspects; interaction of organisms in decomposition processes; decomposition processes as components of whole ecosystems; and modeling of decomposition systems, a single chapter based upon tundra soils. In chapter 12, Fenchel & Harrison trace bacteria-protozoan relationships in aquatic systems and calculate that protozoa can furnish up to one-half the wet weight of the microbes. Elsewhere, protozoa are mentioned as components of decomposer groups. The role of protozoa is speculated upon by Mann on decomposition of marine macrophytes, and Witkamp & Ausmus in forest litter-soil systems.
A protozoan ecologist, however, must possess a sy-nccologic understanding of his subject, and will find many other .chapters with important implications. Thus, the conclusion that most decomposers exist most of the time in a state of suspended animation is important, where the protozoa must likewise encyst until their prey becomes available. Perspective on overall decomposition is provided by Stout et al. and Witkamp, Ausmus & Edwards on soil-litter systems; and Berrie & Saunders on freshwater decomposition. Soil-littc,r systems are dissected by Hissett & Gray into microhabitats, whilc Swift explores species diversity and succession. A recurring theme is that many widely accepted ecologic generalizations are inadequate in the study of particular fields, such as the rhizosphere or decaying leaves in a stream. Deconiposition is not a straightforward process, but involves recycling between microbes and animals, because most biologic systrms contain large amounts of dilute or refractory materials. Understanding the finer detail of decomposition systems is neccssary (as the editors emphasize) if humans wish to manipulate such processes to enhance their own survival. (Floodgate’s chapter on decomposition processes in the sea addresses this idea. ) This is a very valuable book for anyone wishing to learn, or to be brought up to date, about decomposition processes. Each chapter covers its subject in depth, and raises implications for future research. The limited comments on protozoa point out where more work is needed.-STUART S. RAMFORTH, N e w comb College of T u l a n e University, N e w Orleans, L A 70118,