JOURNAL OF BACrERIOLOGY, May, 1965Copyright © 1965 American Society for Microbiology

Vol. 89, No. 5Printed in U.S.A.

Effect of Ethambutol on Nucleic Acid Metabolismin Mycobacterium smegmatis and Its Reversal

by Polyamines and Divalent CationsM. FORBES, N. A. KUCK, AND E. A. PEETS

Experimental Therapeutics Research, Lederle Laboratories Division, American Cyanamid Company,Pearl River, New York

Received for publication 8 January 1965

ABSTRACT

FORBES, M. (Lederle Laboratories Division, Pearl River, N.Y.), N. A. KucK, AND

E. A. PEETS. Effect of ethambutol on nucleic acid metabolism in Mycobacterium smeg-

matis and its reversal by polyamines and divalent cations. J. Bacteriol. 89:1299-1305.1965.-Mycobacterium smegmatis, harvested from cultures inhibited by ethambutol andthen suspended in drug-free medium, exhibited a prolonged lag before growth resumed.Polyamines and magnesium ions shortened this lag. Polyamines and magnesium addedto the culture increased the minimal inhibitory concentration of the drug and reversedthe inhibitory effect of the drug, even when added after the drug had already inhibitedgrowth. When ethambutol was added to a culture in its exponential phase of growth,synthesis of protein and deoxyribonucleic acid (DNA), as measured by incorporationof S5' and p32, continued for 3 hr at a rate slightly less than in the control cells and thenessentially ceased. Synthesis of ribonucleic acid (RNA) was depressed, but it pro-

ceeded even after protein synthesis had ceased. Even though the synthesis of RNAcontinued, the net RNA decreased, and inhibited cells became deficient in RNA. Poly-amines and divalent cations, which reverse the inhibitory effect of the drug, have beenreported to be involved in nucleic acid turnover. These considerations suggested thatethambutol may exert its inhibitory effect by interfering with a function of cellulatpolyamines and divalent cations in RNA metabolism.

Ethambutol (dextro-2, 2'-[ethylenediimino]di-1-butanol) has specific antimycobacterial activityand is therapeutically effective in tuberculosis inanimals (Thomas et al., 1961; Wilkinson et al.,1961) and in man (Bobrowitz, Garber, andSukumalchantra, 1963). Reports on its in vitroand in vivo activity were reviewed by Robsonand Sullivan (1963).

Studies on the mode of action of ethambutolshowed that, even though the drug was taken uprapidly by both proliferating and nonproliferat-ing mycobacteria, it had no effect on the viabilityand metabolism of nonproliferating cells. Whenadded to growing cultures, ethambutol inhibitedgrowth. The inhibitory effect of the drug becameapparent only several hours after addition of thedrug to the culture when the population had atleast doubled (Forbes, Kuck, and Peets, 1962;Kuck, Peets, and Forbes, 1963). Gale and McLain(1963) in studying the effect of ethambutol on thecytology of Mlycobacterium smegmatis found that"absence of probable nuclear substance was themost evident alteration incurred by the cells."The authors postulated that the drug may block

one or more steps in the synthesis of ribonucleicacid (RNA) or deoxyribonucleic acid (DNA),ultimately causing cessation of cellular division.

Preliminary observations (Forbes, Kuck, andPeets, 1964) showed that M. smegmatis, harvestedfrom cultures inhibited by ethambutol and thensuspended in drug-free medium, exhibited a pro-longed lag before growth resumed. In a con-tinuation of these studies, it was found thatpolyamines and divalent cations reversed theinhibitory effect of ethambutol. The present reportdescribes the reversal of the inhibitory effect ofethambutol and the effect of the drug on nucleicacid and protein metabolism in lM. smegmatis.

MATERIALS AND METHODSM. smegmatis ATCC 607 cultured in Sauton's

medium containing 0.02% Tween 80 (polyoxy-ethylene sorbitan monooleate; Atlas Powder Co.,Wilmington, Del.) was used throughout thesestudies. The cultures were incubated at 37 C on areciprocal shaker, and growth was followed bymeasuring optical density at 600 mju in colori-metric tubes (2.54 cm) with a Bausch & LombSpectronic-20 colorimeter. In some experiments,

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the cells were washed and suspended in a nitro-gen-free "resting" medium made with the sameingredients as those of Sauton's medium, exceptthat asparagine was omitted, and the ferric am-monium citrate was replaced with ferric citrate.The number of viable units was estimated by

plating dilutions of culture in Blood Agar Base(BBL). Colony counts were made after 4 to 5 daysof incubation to allow for the slower growth of cellsfrom ethambutol-inhibited cultures.For the determination of protein and nucleic

acid content, cells were harvested by centrifuga-tion at 4 C, washed with water, and proc-essed according to the method of Winder andO'Hara (1962). In this procedure, the cells weresuccessively extracted with cold acetone, cold tri-chloroacetic acid, ethyl alcohol, and hot ethyl alco-hol-ether. The residue was digested at room tem-perature with 1 N KOH, and a sample of the digestwas removed for determination of protein by themethod of Lowry et al. (1951). The remainder ofthe digest was precipitated with perchloric acid,and cold trichloroacetic acid was then added to aconcentration of 5%. RNA was determined fromthe ultraviolet absorption of the supernatantfluid and washes of the precipitate. DNA in theprecipitate was solubilized with hot trichloro-acetic acid and determined by its ultravioletabsorption. Absorption was measured at 268.5rnm, in a Beckman model DU spectrophotometer.RNA and DNA (both obtained from Calbiochem)were used as standards. Human albumin, fractionV (Mann Research Laboratories, New York, N.Y.)was the standard for the protein determination.Ethambutol was determined by an agar diffu-

sion technique with modified Sauton's agar seededwith M. smegmatis (Place and Thomas, 1963).

Protein synthesis was followed by measuringthe S35 (added to the cultures as H2S3504) in theKOH digest of the trichloroacetic acid-insolublefraction of the cells. Nucleic acid synthesis wasfollowed by adding p32 (as H3P3204) to the cul-tures. The p32 incorporated into the RNA andDNA was determined from the difference betweenthe radioactivity of the appropriate cell fractionsbefore and after adsorption of the nucleotidesonto Norit charcoal (Crane, 1958; Harold, 1960).Ultraviolet-absorption measuremenits on the char-coal filtrates indicated that 95 to 100% of thenucleotides were adsorbed onto the charcoal. TheS35 radioactivity in neutralized protein fractionswas determined in a Packard Tri-Carb liquidscintillation spectrometer by use of a naphthalenephosphor solvent system (Werbin, Chaikoff, andImada, 1959). The SI' radioactivity was expressedin absolute units (disintegrations per minute,dpm), the absolute activity being determined bymeans of an internal standard technique (David-son and Feigelson, 1957). The P32 radioactivity wasdetermined in a scintillation well counter (Baird-Atomic Inc., Cambridge, Mass.).

All materials added to the cultures were dis-solved in Sauton's medium. The pH of the stocksolutions was adjusted to 7.2 to 7.4. To test the

effect of cations, the following salts were used:NaCl, Na2SO4, MgSO4, MnSO4.4H2O, ZnSO4-7H20, CUS04, CaHPO4, CaClg, and FeCl3.These stock solutions were autoclaved, and allother materials were sterilized by filtrationthrough ultrafine sintered glass. SpermidiinePO4-6H20 and spermine PO4-6H20 were obtainedfrom Mann Research Laboratories, Inc.; 1,2-diaminoethane, from Matheson Co., Inc., EastRutherford, N.J.; 1,3-diaminopropane hydro-chloride, from Aldrich Chemical Co., Inc., Mil-waukee, Wis.; 1,4-diaminobutane 2HCl (putres-cine), from Distillation Product Industries,Division of Eastman Kodak Co., Rochester, N.Y.;1 ,5-diaminopentane 2HCl (cadaverine) and theamino acids, from Nutritional BiochemicalsCorp., Cleveland, Ohio. Purines, pyrimidines, andnucleosides were obtained from Schwarz BioRe-search Inc., Mount Vernon, N.Y. S35 and P82carrier-free, were obtained from Oak Ridge Na-tional Laboratories, Oak Ridge, Tenn.

RESULTSReversal of ethambutol effect by polyamines and

divalent cations. NVhen growing cells of Ill. smeg-matis were exposed to ethambutol for 3 to 6 hr

PRIOR FTIIANtBPTOIcEXPOSI:,RE AT 4CPTTI

(A.B) Pi't(lR FTHiAVt'Tl

0. a

FIG. 1. Effect of spermidine and magnesium onthe ethambutol-induced lag of Mycobacterium smeg-matis. Ethambutol (10,pg/ml) was added to a culturein its exponential phase of growth (OD600 = 0.20),and the culture was then divided into two parts. Onepart was incubated at 37 C, the other at 4 C. Sixhours after addition of the drug, the cells were har-vested, washed with Sauton's medium, suspended inSauton's medium with and without added spermi-dine (0.5 mM) or MgSO4 , and reincubated at 37 C.The concentration of magnesium in Sauton's me-dium was 4 mM; where magnesium sulfate wasadded, the final concentration was 14 mM. The sus-pensions at 0 hr contained about 2 X 106 viableunits per ml. Counts of viable units showed thatincreases in optical density corresponded to in-creases in cell population. Curves A and F, noaddition; curves B and C, spermidine + MgSO4;curve D, MgSO4 ; curve E, spermidine.

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EFFECT OF ETHAMBUTOL IN M. SMEGMATIS

and then harvested and suspended in drug-freemedium, there was a 20-hr lag before growth re-

sumed (Fig. 1, curve F). In contrast, cells fromcultures to which ethambutol had also beenadded, but which were held at 4 C, exhibited no

prolonged lag before growing when they were

suspended in the drug-free medium (Fig. 1, curve

A). Isotopic measurements showed that bothproliferating (37 C) and nonproliferating (4 C)cells bound C14-ethambutol to the same extent.The prolonged lag, therefore, appeared not to berelated to residual ethambutol in the cells, butrather to the time required for the cells to re-

cover from the damage incurred during growthin presence of the drug.A number of compounds, including amino acids,

purines, pyrimidines, and nucleosides, were testedfor their ability to shorten the lag phase of cellsharvested from ethambutol-inhibited culturesand suspended in drug-free medium. Of the vari-ous materials tested, only certain diamines andpolyamines and magnesium ions had such an

effect. The effect of spermidine, magnesium ions,or both, on the recovery of ethambutol-inhibitedcells suspended in drug-free medium is shown inFig. 1 (curves C, D, and E). These substanceshad no stimulatory effect on the growth in drug-free medium of cells harvested from cultures towhich ethambutol had been added, but whichhad been held at 4 C (Fig. 1, curve B). Otheramines which aided the recovery of ethambutol-inhibited cells were 1,3-diaminopropane, putres-cine, cadaverine, and spermine. No concentrationof spermidine and magnesium was found whichreduced the extent of the lag phase to that oc-

curring in normal cells.Spermidine and magnesium ions also counter-

acted the inhibitory effect of ethambutol in thepresence of the drug. Thus, when spermidine andmagnesium, or both, were added to the medium,the minimal inhibitory concentration (MIC) ofethambutol needed to inhibit growth of M. smeg-matis was increased (Table 1). Assay of theethambutol concentration at the termination ofthe test showed that the culture tubes with addedspermidine and magnesium contained as muchethambutol as the corresponding control tubesto which no additions had been made. Thus, theblocking of the inhibitory action of ethambutolwas not a result of the inactivation of ethambutolby spermidine and magnesium.That the increase in the MIC of the drug was,

indeed, due to the added magnesium ions was

demonstrated by the fact that both MgCl2 andMgSO4 were effective, and equivalent molarconcentrations of NaCl and Na2SO4 had no effect.Addition to the medium of CaCl2 (10 mM) alsoincreased the MIC of ethambutol, but CuS04,

TABLE 1. Effect of added spermidine and magne-sium on the MIC of ethambutol for Mycobacterium

smegmatis in Sauton's medium

Addition to Sauton's mediumMIC of ethambutol*

Spermidine Magnesium sulfate

smoles/ml j.umoles/ml Ug/mlO 0 40 10 160 100 641 0 161 10 321 100 64

10 0 810 10 3210 100 64

* Minimal concentration of ethambutol pre-venting pellicle formation for 72 hr in stationaryculture tubes containing twofold dilutions ofethambutol. In control cultures without drug,pellicle growth was apparent in 24 hr. The con-centration of magnesium in Sauton's medium was4 ,umoles/ml.

ZnSO4, and MnSO4 at concentrations of 1 mmwere ineffective. Ferric chloride was ineffectiveat 10 mm. Higher concentrations of these sub-stances either inhibited growth or caused a pre-cipitate. Cadaverine and putrescine, at con-centrations of 1 and 5 mm, had little effect inincreasing the MIC. Spermine, at 1 and 0.2 mM,had an effect similar to spermidine at 1 mm.

Spermidine and magnesium reversed the in-hibitory effect of the drug, even when added afterthe drug had already inhibited growth. Whenspermidine and magnesium were added to cul-tures 6 hr after ethambutol, they allowed growthto resume in presence of the drug (Fig. 2). Spermi-dine added to the medium at a concentration of0.5 mm gave optimal effects, and magnesium, inconcentrations of 20 to 40 mm, gave maximaleffects in reversing an inhibition of growth by0.05 mM ethambutol. Combinations of spermidineand magnesium were more effective than eitherof the substances alone. In other experiments,it was shown that the ability of these concentra-tions of spermidine and magnesium to reverse theinhibitory effect of ethambutol decreased as theconcentration of the drug was increased.

Effect of ethambutol on nucleic acids and protein.When ethambutol was added to a culture of 31.smegmatis in its exponential phase of growth, thenet protein and DNA continued to increase forthe first 3 hr. Thereafter, the protein remainedconstant, and the DNA continued to increase,but at a sharply reduced rate. The net RNA alsoincreased for the first 3 hr after addition of the

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FORBES, KUCK, AND PEETS

SPERMIDINEplus Mg'

NO ADDITION

u | SPERMIDINE, Mg added

0.2

ETHaMBuTOL

0.1 . . . J|. 8 16 24 32 40

HOURS

FIG. 2. Reversal of ethambutol-inhibition ofMycobacterium smegmatis by spermidine or mag-nesium. Ethambutol (13 ,ug/ml = 0.05 mM) wasadded to a culture in its exponential phase of growth,and the culture was then reincubated at 37 C. Sixhours after addition of the drug, the culture wasdivided into 9-ml volumes. A 1-ml amount of Sau-ton's medium or Sauton's medium containingspermidine or MgSO4, or both, was added to thecultures which were reincubated at 37 C. Spermidinewas added to give a final concentration of 0.5 mm.Concentration of magnesium in Sauton's mediumwas 4 mM; where MgSO4 was added, the final con-centration was 24 mM. Counts of viable units showedthat increases in optical density corresponded toincreases in cell population.

drug, although at a slower rate than the DNA orthe protein; thereafter, in contrast to the DNAand the protein, the net RNA decreased (Fig. 3).Repeated experiments showed that, in growingcells exposed to ethambutol, the protein-dryweight ratio remained constant, the DNA-pro-tein ratio increased slightly, and the RNA-proteinratio decreased steadily (Fig. 4).

Synthesis of protein, as measured by incorpora-tion of S35 into the protein of the cells, continuedfor the first 3 hr after addition of ethambutol tothe culture. Thereafter, Ino further enrichment inradioactivity in the protein occurred, indicatingthat protein synthesis had ceased (Fig. 3). Syn-thesis of DNA, as measured by incorporation ofp32 into the DNA of the cells, continued for thefirst 3 hr after addition of ethambutol at a rateonly slightly less than in the noninhibited cells.Thereafter, incorporation of p32 into the DNAcontinued, but at a sharply reduced rate (Fig. 3).Incorporation of p32 into RNA in the presence of

PRGTEIN

//3

I 3 6 9

8

21

DNA

/

/,_//

6 , 9

o 3 6 9

RNA

3 6 9

0 3 6 9 0 3 6 9 0 3 6 9

HOUR HOUR HOUR

FIG. 3. Effect of ethambutol on protein, DATA,and RNA in Mycobacterium smegmatis. p32 (finalconcentration, 0.33 Ac/ml) or S35 (final concentra-tion, 0.018 iuc/ml) was added to a culture in theearly exponential phase (OD600 = 0.20) of growth.The culture was then immediately divided into twoparts; ethambutol (10 Ag/ml) was added to one andboth parts were reincubated. Samples of culturewere collected at intervals, and protein and nucleicacid contents of the cells and radioactivity in thecell fractions were determined. At 0 hr, the culturescontained per 100 ml; protein, 2,500 ,ug; RNA, 940,ug; DNA, 270 Ag. Symbols: solid lines, control;broken lines, ethambutol.

ethambutol proceeded at a slower rate than inthe noninhibited cells, but continued even afterprotein synthesis had ceased. Even though theRNA synthesis in the inhibited cells continued,the net RNA decreased (Fig. 3).A decrease in the net RNA indicates that the

rate of degradation exceeds the rate of synthesis.To test whether the drug blocked steps in thesynthesis of RNA, precursors of nucleic acidswere added to the cultures. Adenine, guanine,uracil, cytosine, thymine, and nucleosides, in con-centrations of 10 or 100lOg/ml, did not counteractthe inhibitory effect of ethambutol. To testwhether the drug accelerated enzymes degradingRNA, cells were suspended in a nitrogen-free"resting" medium. In this medium, the RNA of

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EFFECT OF ETHAMBUTOL IN M. SMEGMATIS

PROTEIN/DRY WEIGHT

. I I.

DNA/PROTEIN

0

H

HOURS

FIG. 4. Effect of ethambutol on the protein andnucleic acid composition of Mycobacterium smeg-matis. A culture in the early exponential phase ofgrowth (ODro = 0.20) was divided into two parts;ethambutol (10 ,g/ml) was added to one, and bothparts were reincubated. Cells were collected at inter-vals and freeze-dried. Analyses were performed on

weighed samples of dried cells. The vertical barsrepresent the range of values of duplicate determina-tions in three replicate experiments. Symbols: solidlines, control; broken lines, ethambutol.

the cells gradually decreased and then remainedat a constant level. Ethambutol did not affectthe rate of degradation (Fig. 5).

DISCUSSIONInhibition of growth of MI. smegmatis by etham-

butol was accompanied by a decrease in the RNA

LiOURS

FIG. 5. Effect of ethambutol on RNA and DNAin Mycobacterium smegmatis cells in a nitrogen-freeSauton's medium. Cells from a culture in its ex-

ponential phase of growth were harvested, washedwith nitrogen-free Sauton's medium, and suspendedin this medium with or without ethambutol (10,g/ml). The suspensions were incubated at 37 C,cells were collected at intervals, and protein andnucleic acid contents were determined. Symbols:solid lines, control; broken lines, ethambutol.

content of the cells. During the first 3 hr afteraddition of the drug to the culture, RNA metabo-lism was depressed more than DNA or proteinmetabolism, and the fall in the RNA-proteinratio preceded the arrest of growth (Fig. 3 and 4).A deficiency in RNA would result in cessation ofprotein synthesis and growth. The experiments donot permit a conclusion as to whether the RNAdeficiency is due to a depression of the synthesisof RNA coupled with a normal rate of breakdown,or to instability of the RNA formed in presenceof the drug. No indication was found that etham-butol accelerated the degradation of RNA byenzymes (Fig. 5).

Precursors of the nucleic acids did not relievethe growth inhibition by ethambutol, but poly-

0.4

0.3

0.21-

0.31-

0.21-.

0.1

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amines and metal cations did. These substancesdid not inactivate the drug. One possible explana-tion of their effect may be that ethambutol actsby interfering with a function of cellular poly-amines and metal cations. The polyamines andmetal cations added to the medium would thencounteract this action of the drug. This possi-bility is supported by the fact that ethambutol isa substituted ethylenediamine with structuralsimilarity to polyamines, and that alkanol-substituted ethylenediamines, such as etham-butol, are known to form metal chelates (Hall,Dean, and Pacofsky, 1960). Polyamines anddivalent cations have been reported to play arole in nucleic acid turnover in bacteria (Taborand Tabor, 1964). They stabilize ribosomes(Cohen and Lichtenstein, 1960; Martin and Ames,1962), inhibit RNA degradation in whole cells(Herbst and Doctor, 1959), and are required forthe maximal synthesis of RNA by RNA poly-merase (Doerfler et al., 1962; Krakow, 1963; Foxand Weiss, 1964). In these reactions, it is oftenfound that there is a concentration of the poly-amine that is optimal, higher concentrationsbeing less effective. Metal cations, specificallymagnesium or calcium, may often replace poly-amines in their functional role in nucleic acidturnover. It is characteristic that higher con-centrations of the metal ions than of the poly-amines are needed to accomplish the same effect(Tabor and Tabor, 1964). The pattern of theeffect of spermidine and magnesium in counter-acting ethambutol, thus, shows analogies withthe effect of these substances in nucleic acidmetabolism.The data show that ethambutol has no effect

on nonproliferating cells of MI. smegmatis. Thedrug, after a delay, arrests the growth of activelyproliferating cells. Cytological examinations(Gale and McLain, 1963) of cells inhibited byethambutol revealed no alteration of the integrityof the cell wall and membrane. Chemical deter-minations showed that inhibited cells becamedeficient in RNA. The early effects of the drugare reversible. Cells harvested from cultures in-hibited by ethambutol and then suspended indrug-free medium eventually resume growth.Polyamines and divalent cations, which areknown to be involved in nucleic acid turnoverand the maintenance of the integrity of ribo-somes, aid the recovery of these cells. Thesesubstances in the presence of the drug alsocounteract the inhibitory effect of the drug. Allthese considerations are consistent with the viewthat ethambutol exerts its inhibitory effect ongrowth by interfering with a function of cellularpolyamines and divalent cations in the synthesisor stabilization of RNA.

LITERATURE CITEDBOBROWITZ, I. D., M. GARBER, AND Y. SUKU-MALCHANTRA. 1963. The use of ethambutol inpulmonary tuberculosis. Trans. Res. Conf.Pulmonary Diseases, 22nd, Washington, D.C.,p. 254-261.

COHEN, S. S., AND J. LICHTENSTEIN. 1960. Poly-amines and ribosome structure. J. Biol. Chem.235:2112-2116.

CRANE, R. K. 1958. Use of charcoal to separatemixtures of inorganic, ester, and nucleotidephosphates. Science 127:285-286.

DAVIDSON, J. D., AND P. FEIGELSON. 1957. Prac-tical aspects of internal-sample liquid scintilla-tion counting. Intern. J. Appl. Radiation Iso-topes 2:1-18.

DOERFLER, W., W. ZILLIG, E. FUCHS, AND M.ALBERS. 1962. Untersuchungen zur Biosyntheseder Proteine V. Z. Physiol. Chem. 330:96-123.

FORBES, M., N. A. KucK, AND E. A. PEETS. 1962.Mode of action of ethambutol. J. Bacteriol.84:1099-1103.

FORBES, M., N. A. KUCK, AND E. A. PEETS. 1964.Studies of the mode of action of ethambutol.Trans. Intern. Congr. Chemotherapy, 3rd,Stuttgart, W. Germany, p. 174-177.

Fox, C. F., AND S. B. WEISS. 1964. Enzymaticsynthesis of ribonucleic acid. II. Properties ofthe deoxyribonucleic acid-primed reaction withMicrococcus lysodeikticus ribonucleic acid poly-merase. J. Biol. Chem. 239:175-185.

GALE, G. R., AND H. H. MCLAIN. 1963. Effect ofethambutol on cytology of Mycobacteriumsmegmatis. J. Bacteriol. 86:749-756.

HALL, J. L., W. E. DEAN, AND E. A. PACOrSKY.1960. Metal chelates of alkanol-substitutedamines. J. Am. Chem. Soc. 82:3303-3308.

HAROLD, F. M. 1960. Accumulation of inorganicpolyphosphate in mutants of Neurospora crassa.Biochim. Biophys. Acta. 45:172-188.

HERBST, E. J., AND B. P. DOCTOR. 1959. Inhibitionof ribonucleic acid degradation in bacteria byspermine. J. Biol. Chem. 234:1497-1500.

KRAKOW, J. S. 1963. Ribonucleic acid polymeraseof Azotobacter vinelandii. III. Effect of poly-amines. Biochim. Biophys. Acta 72:566-571.

KUCK, N. A., E. A. PEETS, AND M. FORBES. 1963.Mode of action of ethambutol on Mycobac-terium tuberculosis, strain H37Rv. Am. Rev.Respirat. Diseases 87:905-906.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem.193:265-275.

MARTIN, R. G., AND B. N. AMES. 1962. The effectof polyamines and of poly U size on phenyl-alanine incorporation. Proc. Natl. Acad. Sci.U.S. 48:2171-2178.

PLACE, V. A., AND J. P. THOMAS. 1963. Clinicalpbarmacology of ethambutol. Am. Rev. Re-spirat. Diseases 87:901-904.

RoBSON, J. M., AND F. M. SULLIVAN. 1963. Anti-tuberculosis drugs. Pharmacol. Rev. 15:169-223.

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THOMAS, J. P., C. 0. BAUGHN, R. G. WILKINSON,AND R. G. SHEPHERD. 1961. A new syntheticcompound with antituberculous activity inmice: ethambutol (dextro-2,2'-(ethylenedi-imino)di-1-butanol). Am. Rev. Respirat. Dis-eases 83:891-893.

WERBIN, H., I. L. CHAIKOFF, AND M. R. IMADA.1959. RaDid sensitive method for determining

H3 water in body fluids by liquid scintillationspectrometry. Proc. Soc. Exptl. Biol. Med.102:8-12.

WILKINSON, R. G., R. G. SHEPHERD, J. P. THOMAS,AND C. 0. BAUGHN. 1961. Stereospecificity in anew type of synthetic antituberculous agent.J. Am. Chem. Soc. 83:2212-2213.

WINDER, F. G., AND C. O'HARA. 1962. Effects ofiron deficiency and of zinc deficiency on thecomposition of Mycobacterium smegmatis. Bio-chem. J. 82:98-107.

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