36 J . PROTOZOOL., VOL. 30, NO. I , FEBRUARY 1983

15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. 1951. Protein measurement with the Fohn phenol reagent. J . Biol. Chem., 193: 265-275.

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17. Majumder, G. C. & Turkington. R. W. 197 1. Adenosine 3’,5’- monophosphate-dependent and -independent protein phosphokinase isoenzymes from mammary gland. J . Biol. Chem.. 246: 2650-2657.

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198 1. Tetrahymena calcium binding protein is indeed a calmodulin. J . Biochem., 89: 333-336.

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10: 311-316.

Received 26 III 82; accepted 2 VII I 82

J Prorozoul 30(1), 1983. pp 36-40 c I983 b) the Society of Protoroologists

Enzymes of de novo Pyrimidine Biosynthesis in Babesia rodhaid JOHN W. HOLLAND, ANNETTE M. GERO, and WILLIAM J. OSULLIVAN

School y / Biochemisiry. 1’niversii.v of New South Wales, Kensingion. N.S. W., Australia 2033

ABSTRACT. The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2); dihydroorotase (DHOase: E.C. 3.5.2.3); dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5’-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO- DHase, OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacety1)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent K, of 7 pM. Dihydro-5-azaorotate inhibited the DHO-DHase (K,, 16 pM) and Sazaorotate (K,, 21 pM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (K,, 0.3 pM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product, UMP (K,, 120 pM) and the purine nucleotide. XMP (K,. 95 rM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.

N c o m m o n with m a n y other parasitic protozoa, Babesia spp. I require a supply o f pyrimidines for reproduction and growth. I t appears that some strains o f Babesia preferentially obtain pyrimidines by de novo synthesis of U M P rather than salvage synthesis o f preformed pyrimidines f rom the host erythrocyte. Thus. Irvin a n d co-workers ( 1 7 , 18) reported that two rodent strains (B . microfi a n d B. rodhaini) incorporated labeled uridine

’ We should like to thank Dr. Graham F. Mitchell for supplying the parasite strain and Mr. K. G. Finney for the generous gifts of PALA and [IT]-carbamylphosphate. This work was supported in part by the National Health and Medical Research Council of Australia.

a n d thymidine into parasite nucleic acid but, by contrast , neither o f t w o bovine strains tested ( B . divergens and B. major) showed a n y significant incorporation. T h e implication of these results is that the rodent strains rely o n salvage for their pyrimidine requirements, whereas de novo synthesis is more important i n the bovine strains. However, t he incorporation studies were complicated by the fact t ha t t he experiments were performed using cultured infected erythrocytes ra ther than isolated para- sites. Further, n o direct enzyme studies were carried o u t o n the isolated parasites.

Recently, direct evidence for t he presence of the de n o v o pathway in a rodent strain, B. hylomysci, was obtained by Gero

HOLLAND ET AL.-PYRIMIDINE BIOSYNTHESIS IN BABESIA 37

& Coombs ( 1 0), who demonstrated the presence of the last four enzymes of the pathway. To our knowledge this is the only reported observation of these enzymes in Babesia. We report here the detection, some preliminary characterizations and the inhibition of enzymes of the pathway of de novo pyrimidine biosynthesis in another rodent species, B. rodhaini.

MATERIALS AND METHODS Materiak All materials were purchased from Sigma Chemical

Co. and were of the highest analytical purity. N-(phosphon- acety1)-L-aspartate (PALA) was synthesised by K. G. Finney in this laboratory by the procedure of Collins and Stark (5). Oxi- purinol, allopurinol, and menoctone were a gift from Burroughs Wellcome Australasia and dihydro-5-azaorotate a gift from the Wellcome Research Laboratories, Research Triangle Park, NC.

Radiochemicals. Na[I4C]HCO, (0.1 mCi/mmol) was obtained from the Radiochemical Centre. New England Nuclear Corp. provided [~a rboxy-~~C]oro t i c acid (4 1.25 mCi/mmol), [4- 14C]orotic acid (9.36 mCi/mmol) and [~arboxy-~~C]orotid- ine-5'-phosphate (OMP) (34.9 mCi/mmol). [14C]Carbamyl phosphate (0.024 mCi/mmol) was a gift from K. G. Finney. ~-5,6-[4-(~C]- and ~-5,6-[carboxy-~~C]dihydroorotic acid ([4-I4C]- or [~arboxy- '~C]~-DHO, respectively) were prepared from [4- I4Cc]- and [carboxy-i4C]orotic acid respectively, as described by Smithers et al. (29).

Parasites. Babesia rodhaini Van den Berghe et al., 1950, was obtained from Dr. G. F. Mitchell of the Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. The strain was maintained in female white balb/c mice by a bi-weekly intraperitoneal passage of 1 O8 parasited20 g mouse. Mice treat- ed in this way developed a parasitaemia of 50% four days after inoculation. Routine staining and counting of blood smears showed that significant levels of reticulocytosis occurred if the parasitaemia was allowed to increase beyond this level.

Parasites were isolated from the infected erythrocytes as de- scribed previously for the isolation of P. berghei (1 1). The iso- lated parasites were ruptured by sonication. Initial enzyme as- says were carried out on whole homogenates. In some cases the homogenate was ultracentrifuged at 100,000 g for 1 h at 4°C and the supernatant or pellet used as the enzyme source. As the isolated parasites are encompassed by collapsed erythrocyte membranes, normal mouse erythrocytes were subjected to an identical isolation procedure so that possible contamination by host cell enzymes could be determined.

Protein concentrations were determined by the method of Itzhaki & Gill (1 9), using bovine serum albumin as the standard.

Enzyme assays. Carbamylphosphate synthetase (CPS 11: E.C. 6.3.5.5) activity was measured by the conversion ofNa[14C]HC0, to [ ''C]carbamylaspartate using endogenous aspartate transcar- bamylase (ATCase, E.C. 2.1.3.2) as the coupling enzyme (22). The standard assay system contained N-2-hydroxyethylpiper- azine-N'-2-ethane sulfonic acid (HEPES)-KOH (pH 7.4) 50 mM, ATP 25 mM, MgC1, 100 mM, L-glutamine 3.3 mM, dithio- threitol 1 mM, L-aspartate 10 mM, Na[14C]HC0, (0.1 mCi/ mmol) 16.7 mM, and 0.4 mg of protein in a total volume of 0.6 ml. The assay mixture, in a 20 ml scintillation vial, was incubated at 30°C for 30 rnin and the reaction terminated by the addition of 0.06 ml of 5 M formic acid. The vials were transferred to an oven at 1 10°C and evaporated to dryness. The residue was redissolved in 1 ml of water and 10 mls of 0.5% PPO in to1uene:triton X-100 2:l (v/v) was added. The vials were counted in a quench corrected Packard Tricarb scintillation counter (Model 2650).

Aspartate transcarbamylase (ATCase) (E.C. 2.1.3.2) activity was measured by the incorporation of radioactivity from [ 14C]carbamylphosphate into carbamylaspartate (22). The stan-

TABLE I . Activities ofpyrimidine biosynthetic enzymes in Babesia rod- haini.a

Enzyme activities (nmoles/h/mg protein) Uninfected Plas-

Babesia erythrocyte Babesia modium Enzymes rodhaini membrane hylomisci berghei

CPS I1 2 b 0 n.d.' 6 ATCase 454 * 54 (12) 0 n.d. 1044 DHOase 41 -t 8 (4) 0.06 9 378 DHO-DHase 93 k 19 (4) 0.3 17 12 OPRTase 7.8 -t 2.9 (10) 0.2 5.3 96 ODCase 22 * 5 (16) 0.2 n.d. 162

~~

a Enzyme activities obtained for B. rodhaini are compared to those for mouse eyrthrocytes carried through the same procedure as for the isolation of parasites and with published values for B. hyiomisci (10) and for P. berghei (1 3).

A single determination only was obtained for CPS 11. For the other enzymes the number of estimations have been indicated in parentheses and the standard errors shown.

Not determined.

dard assay system contained tris-(hydroxymethy1)-amino meth- ane (TR1S)-HCl (pH 8.5) 100 mM, L-aspartate 10 mM, ['"CI- carbamylphosphate (0.024 mCi/mmol) 0.2 mM and 0.1 mg of protein in a total volume of 1 .O ml. The reaction was initiated by the addition of ['4C]carbamylpho~phate and carried out at 30°C for 30 min.

Dihydroorotase (DHOase: E.C. 3.5.2.3) was assayed in the reverse direction (the conversion of dihydroorotate to carba- mylaspartate) essentially as described by Finney & O'Sullivan (6). The assay system contained TRIS-HCl (pH 8.5) 100 mM, [4-I4C]-~-DHO (0.72 mCi/mmol) 0.5 mM and 0.1 mg of protein in a total volume of 0.2 ml. The reaction was carried out for 30 rnin at 30"C, stopped with 0.02 ml of 5 M formic acid and the reactants separated by electrophoresis (30 min at 6 kV) on Whatman 3 MM chromatography paper. DHOase activity was determined from the percentage of total radioactive counts as- sociated with each ofthe carbamyl aspartate, L-DHO, and orotic acid spots.

Dihydroorotate dehydrogenase (DHO-DHase, E.C. 1.3.3.1) was assayed by a coupled radioassay using excess yeast orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10) and oro- tidylate decarboxylase (ODCase: E.C. 4.1.1.23) to convert [car- boxy-I4C]-~-DHO to UMP and 14C0, (29). The standard assay system contained HEPES-KOH (pH 8.0) 10 mM, MgCI, 7.5 mM, 5 -phosphoribosyl- 1 -pyrophosphate (P- Rib-PP) 0.2 5 mM, [ c a r b ~ x y - ~ ~ C ] - ~ - D H O (0.2 mCi/mmol) 0.06 mM, OPRTase- ODCase (mixed enzyme from yeast) 2 mIU and 0.2 to 0.25 mg of protein in a total volume of 1.0 ml. Reactions were carried out for 30 rnin at 30°C in 20 ml scintillation vials sealed with rubber Suba seals. I4CO2 liberated from the reaction was col- lected in hyamine hydroxide (0.1 ml) and counted as described previously (8).

OPRTase activity was assayed in a similar manner to DHO- DHase, using endogenous ODCase as the coupling enzyme and measuring the release of 'TO, (8). The standard assay system contained HEPES-KOH (pH 8.0) 50 mM, P-Rib-PP 0.25 mM. MgCI, 5 mM, [~arboxy-'~C]orotic acid (0.24 mCi/mmol) 0.2 mM and 0.2 to 0.25 mg of protein in a total volume of 1 .O ml. Reactions were carried out at 30°C for 30 min.

ODCase activity was measured by the release of I4CO2 from [~arboxy-~~C]OMP during its conversion to UMP (8). The stan- dard assay system contained HEPES-KOH (pH 8.0) 50 mM. [~arboxy- '~C]OMP (0.94 mCi/mmol) 0.02 mM and 0.1 mg of

38 J . PROTOZOOL.. VOL. 30, NO. I . FEBRUARY 1983

T ~ H I E 11. Summary of kinetic data and inhibrtion constants.for the B. rodhaini pyrimidine hios.vniheric enzymes."

K,,, K, ~~~ ~~

.4 TCase Aspartate 1.9 mM - Carbamyl phosphate 12 ( k 2 ) p M - PALA - 7 ( i 3 . 6 ) pM

DHO- DHase L-DHO 15.4 (k9.0) pM - Di hydro- 5 -azaorotate - 16.1 (+9.I)pM

OPRTasc Orotate 3.8 (T0.8) pM - 5-Azaorotate - 4.7 (k2 .2) pM

OMP 0.77 (k0.03) pM - UMP - I20 (?60) pM XMP - 95(+15)pM 6-Aza-UMP - 0.27 (k0.14) pM

Apparent K, values and inhibition constants (K,) for the most ef- fective inhibitors tested, were determined from weighted regression pro- cedures (4). Standard errors obtained from these analyses are included in parentheses, except for the value for aspartate in the ATCase reaction. The substrate inhibition observed for this substrate made the statistical treatment of doubtful validity. and the K, was estimated by graphical procedures. All inhibitors exhibited competitive inhibition with respect to the substrate.

ODCase

protein in a total volume of 1 .O ml. Reactions were carried out for 30 min at 30°C.

For kinetic and inhibition studies of ATCase, DHO-DHase. OPRTase and ODCase, the standard assay conditions were al- tered with respect to substrate. inhibitor concentrations. and reaction times. The linearity with respect to time and protein concentration was established for all assays. None of the inhib- itors tested had any significant effect on the respective coupling enzymes under the assay conditions reported. Kinetic data were fitted to the appropriate rate equations using the computer pro- grams described by Cleland (4).

RESULTS Activities of Pyrimidine de now Biosj-nthetic. Enzjwies

The activities of the six enzymes of pyrimidine biosynthesis de novo, CPS 11. ATCase. DHOase, DHO-DHase, OPRTasc, and ODCase. were demonstrated in B. rodhaini homogenates (Table I). The result for CPS I1 represents a single determination only. Attempts were made to duplicate this result but the en- zyme appeared to be very labile, and the counts obtained were not significantly above background.

As isolated, the parasites are encapsulated in host red cell membrane. Thus, the results are compared to estimates of the enzyme activities in the membrane material obtained from nor- mal mouse erythrocytes when they were subjected to the same procedure as used for the preparation of the parasites. These estimates indicated minimal contribution to the parasitic en- zymatic activities by host cell enzymes. Activities of the en- zymes from B. hylorqsci (10) and from the rodent malarial parasite, Plasn~odiiirn herghei ( 1 3). are included for comparison in Table I .

As for all systems reported so far, the highest activity was observed for ATCase. The activity of DHO-DHase was also shown to be five to six times higher than that reported for other parasitic sporozoa (10. 13). As observed in other systems (8. 20, 37), the ODCase activity was significantly higher than the OPRTase.

Kinetic and Inhihition Studies Four of the enzymes were investigated in further detail. A

summary of the kinetic data obtained is collected in Table 11, together with inhibition constants of the most effective inhibi- tors. .4 TCase. Apparent K,,, values for both aspartate and carbamyl

phosphate were determined for ATCase. Substrate inhibition at concentrations above 10 mM was observed for aspartate. This has been demonstrated for ATCase from other systems (1 6,27). Inhibition by PALA, an analog of the activated complex for the ATCase reaction, was shown to be competitive, and the appar- ent K, was determined as 7 pM, some 2-3 orders of magnitude higher than reported in other systems (5, 14, 30).

DHO-DHase. This enzyme was found to occur in the 100,000 g pellet. The K,,, value of 15.4 pM for the B. rodhaini DHO- DHase was similar to that reported for the enzyme from B. hylomysci (21 pM) (lo) and from P. berghei (23 pM) (1 1). The most effective inhibitor (competitive with respect to L-DHO) was dihydro-5-azaorotate (K,, 16 pM), an analog of L-DHO which has been shown to inhibit DHO-DHase from Ehrlich Ascites cells (24), P. berghei ( 1 I ) , B. hylomysci (lo), and mouse liver (26). Menoctone (0.3 mM). a ubiquinone analog, was also shown to cause 100n/n inhibition of the Babesia DHO-DHase.

OPRTase. The K,,, value for orotic acid was determined to be 3.8 pM. This is similar to the value of 2 pM reported for the mouse Ehrlich Ascites cells enzyme (27), though lower than values reported for the enzymes from calf thymus (32 pM) (21) and P. berghei (14 pM) (25). The K,, for P-Rib-PP was not determined but no increase in reaction velocity occurred be- tween 0.02 mM and 0.2 mM P-Rib-PP.

A number of pyrimidines and pyrimidine analogs were tested as inhibitors of the OPRTase reaction. Uracil and the uracil analog, 6-azauraci1, at concentrations of 0.4 mM, caused no significant inhibition. The substrate analog, 5-azaorotic acid, was demonstrated to be a competitive inhibitor with respect to orotic acid with a K, of 4.7 pM.

Allopurinol and oxipurinol which, apart from being inhibitors of xanthine oxidase, also inhibit the OPRTase-ODCase se- quence in erythrocytes (S), were tested as inhibitors of OPRTase in B. rodhaini. No measurable effect was observed with allo- purinol (0.4 mM) and only a low level of inhibition (1 6%) was observed with oxipurinol (0.05 mM). These compounds had a similar low inhibitory effect on the enzyme from P. berghei (25).

ODCase. The K,,, for OMP was determined to be 0.77 pM, which i s of the same order as found for human erythrocytes (2), though somewhat lower than values of 2-9 pM reported for other mammalian systems (20). The product of the reaction UMP was a weak inhibitor, 6-aza-UMP a good inhibitor, and the purine nucleotide, XMP, only a moderate inhibitor of the ODCase reaction. The inhibition was competitive for all three compounds. and K, values are included in Table 11. The inhi- bition constants of 6-aza-UMP and UMP are in good agreement with those reported in mammalian tissues ( I , 3, 27). The in- hibition of XMP was less effective in Babesia than in other tissues (3. 9).

Allopurinol and oxipurinol, which inhibit human ODCase (7, 8). did not have any marked inhibitory effect on the enzyme from B. rodhaini. Where allopurinol or oxipurinol was prein- cubated in the assay mixture with added P-Rib-PP and MgCI, to form the respective ribonucleotide, n o increase in inhibition was observed. This probably reflects an insignificant level of formation of the nucleotides rather than a lack of inhibition of ODCase. Similar experiments with P. herghei (25) indicated an apparent increase in ODCase activity, suggesting a possible ag- gregation or stabilization of the OPRTase-ODCase complex. The effect was not observed with B. rodhaini.

HOLLAND E T AL.-PYRIMIDINE BIOSYNTHESIS IN BAEESIA 39

DISCUSSION In this investigation five of the six enzymes of pyrimidine

biosynthesis d e novo, ATCase, DHOase , D H O - D H a s e , OPRTase, and ODCase, were shown conclusively to be present in B. rodhaini for the first time. The single value observed for CPS I 1 is of the same order as reported for the enzyme from P. herghei (1 3), but difficulty was experienced in reproducing this value, presumably due to instability of the enzyme. This was disappointing as this enzyme is a n important control point in the pathway in eukaryotic systems. The mammalian enzyme has been shown to be very unstable (3 l), and it is possible that conditions for stabilization of CPS I1 from B. rodhaini were not achieved in this study.

As reported for all other sources, the highest specific activity was observed for ATCase. Its kinetic behavior appears to be similar to the enzyme from other systems ( 5 , 15, 27, 30). How- ever, the ATCase in crude homogenates from B. rodhaini dif- fered with respect to the relatively high K, value obtained for PALA. PALA has previously been shown to be a potent and specific inhibitor of the enzymes from E. coli (5), mouse spleen (14), and C13/SV cultured hamster cells (30). The K, values reported for the enzymes from these sources are of the order of 1-10 nM, 2-3 orders of magnitude lower than those obtained for the B. rodhaini enzyme (7 pM). PALA is still, however, an effective inhibitor of the B. rodhaini ATCase and may be worthy of consideration as a n antiproliferative drug in Babesia infec- tions.

The enzymes that convert dihydroorotate t o orotate are di- verse in nature, and both soluble and particulate DHO-DHase activities have been reported for protozoa (10, 12, 23). The enzyme in B. hylomysci was shown to be particulate with a specific activity of 17 nmoles/h/mg protein (1 0). The enzyme from B. rodhaini was also particulate and had a specific activity some 5-6 times higher than that found in B. hylomysci and in P. berghei ( I 1, 13). Both dihydro-5-azaorotate and menoctone were shown to be potent inhibitors of the B. rodhaini DHO- DHase. The potential of these compounds as antiparasitic agents may be substantial as DHO-DHase is not functional in the host cell, the mature erythrocyte (1 1, 28).

Irvin & Young (1 7) found that B. rodhaini in infected eryth- rocytes could not incorporate tritiated orotic acid, implying that the parasites lacked the enzymes OPRTase and ODCase. Both activities were, however, demonstrated in B. rodhaini in this investigation.

The apparent K,, for orotic acid of 3.8 pM was similar to that reported for Ehrlich Ascites cells (27 ) . O f the pyrimidines and pyrimidine analogs tested as inhibitors of OPRTase, only the substrate analog, 5-azaorotate, was a n effective inhibitor (K,, 4.7 pM), indicating that control a t this step through inhibition by end products of the pathway is unlikely. However, apart from CPS 11, OPRTase was found to be the enzyme with the lowest specific activity of the five enzymes measured. Shoaf & Jones (27) made a similar observation for this enzyme from Ehrlich Ascites cells and suggested that it may be rate limiting for the pathway in some circumstances, particularly a t relatively low P-Rib-PP levels.

ODCase in B. rodhaini had similar kinetic properties to the enzyme in other systems. The activity was found to be three- fold higher than the OPRTase activity. Similar results have been reported for the two enzymes from human erythrocytes (8) and Ehrlich Ascites cells (27). The apparent K, for O M P of 0.77 pM was comparable to reported values from human tissues (2). Product inhibition by U M P that has been widely noted in other systems (1 ,3 ,27) was also observed for the B. rodhainz enzyme.

In conclusion, B. rodhaini has been demonstrated to contain at least five enzymes of the pyrimidine de novo pathway and

thus to have the potential of synthesizing pyrimidines by this route. The properties of the enzymes indicate that they are sim- ilar t o the enzymes from other eukaryotic sources. Some pre- liminary inhibition studies have been carried out, and the results presented indicate that pyrimidine analogs may possess useful antisporozoan activity through their inhibition of the enzymes of the de novo pyrimidine biosynthetic pathway. It is anticipated that this investigation will provide a basis for a study of the bovine strains of Babesia, B. bovis and B. bigemina. which are of veterinary importance.

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88: 93-103.

Received 14 I V 82; accepted 5 VIII 82

“SHORTER COMMUNICATIONS” NOW ACCEPTED IN THE JOURNAL

We are happy to announce recent Executive Committee approval of a slightly different kind of paper in our Journal, the short communication. Limited t o 1500 words (or 1200 i f a figure and table are included), these concise manuscripts may be expected t o experience more rapid publication, ordinarily, than the typical “full paper” or longer communication. Style, format, etc. must conform to the usual “rules” for all papers: see our “Instructions t o Contributors” in the back of the present issue of the Journal, where a few more details regarding the new category-shorter communications-are also included. Consider sending us a manuscript of this kind! - John 0. Corliss, Managing Editor

FORTHCOMING CONFERENCE ON MALARIA AND BABESIOSIS

The Second International Conference on Malaria and Babesiosis will take place 19-22 September 1983 in Annecy, France. An informative package for the conference is available. Those interested in receiving this packet may address their requests to:

Dr. Micha Roumiantzeff Foundation Merieux 17 Rue Bourgelat 69002 Lyon, FRANCE or Urbana, IL 6 180 1 U.S.A.

Dr. Miodrag Ristic College of Veterinary Medicine University of Illinois

Phone: 838-06-10 Ext. 512

Phone: 21 7/333-267 I

NOTICE REGARDING SLIGHT INCREASE IN PAGE CHARGES

One of the decisions made by the Executive Committee a t the recent meetings of the Society of Protozoologists in San Francisco was t o raise the page-charge fee for Journal papers from $36 to $40 per page. The increase is relatively small; but it brings the author’s (or h idher institution’s) share of the actual cost of producing and printing a page closer to SO%, which seems justified. Many scientific journals charge a much higher fee. Much of the Journal‘s costs are (still) subsidized largely by membership dues and subscriptions. The only alternative to having higher page charges is to publish fewer pages, a result displeasing to members of the Society who would prefer to see us moving in the direction of more pages (and, consequently, more rapid publication of manuscripts). Members-for their $26 (or $1 3, for students) annual dues-still receive up to f i v e j k e pages (see “Instructions to Contributors” in the back of this issue), so this is one way to avoid the problem of page-charge costs if the author is having difficulty in finding sufficient funds to cover them.-John 0. Corliss, Managing Editor