The Metabolism of Tay-Sachs Ganglioside:

Catabolic Studies with Lysosomal Enzymes

from Normal and Tay-Sachs Brain Tissue

JOHNF. TALLMAN, WILLIAM G. JOHNSON,and ROSCOE0. BRADY

From the Developmental and Metabolic Neurology Branch, National Instituteof Neurological Diseases and Stroke, National Institutes of Health,Bethesda, Maryland 20014, and the Department of Biochemistry, GeorgetownUniversity School of Medicine, Washington, D. C. 20007

A B S T R A C T The catabolism of Tay-Sachs ganglioside,N-acetylgalactosaminyl- (N-acetylneuraminosyl) -galac-tosylglucosylceramide, has been studied in lysosomalpreparations from normal human brain and brain ob-tained at biopsy from Tay-Sachs patients. Utilizing Tay-Sachs ganglioside labeled with '4C in the N-acetylgalac-tosaminyl portion or 3H in the N-acetylneuraminosylportion, the catabolism of Tay-Sachs ganglioside may beinitiated by either the removal of the molecule ofN-acetylgalactosamine or N-acetylneuraminic acid. Theactivity of the N-acetylgalactosamine-cleaving enzyme(hexosaminidase) is drastically diminished in suchpreparations from Tay-Sachs brain whereas the activityof the N-acetylneuraminic acid-cleaving enzyme (neu-raminidase) is at a normal level. Total hexosaminidaseactivity as measured with an artificial fluorogenic sub-strate is increased in tissues obtained from patients withthe B variant form of Tay-Sachs disease and it is vir-tually absent in the 0-variant patients. The addition ofpurified neuraminidase and various purified hexosamini-dases exerted only a minimal synergistic effect on thehydrolysis of Tay-Sachs ganglioside in the lysosomalpreparations from the control or patient with the0 variant of Tay-Sachs disease.

INTRODUCTION

Infantile Tay-Sachs disease, the most common of thelipid storage diseases, is a fatal inborn error of ganglio-side metabolism, inherited as an autosomal recessivetrait. The earliest clinical descriptions of the disease

Received for publication 18 February 1972 and in revisedform 1 May 1972.

date fronm the 19th century and over 599 cases have beenreported (1). Onset of the disease is in the first 6 monthsof life and is characterized by apathy, hyperacusis, motorweakness, and appearance of a macular cherry-red spotin the retina. Seizures and progressive mental deteriora-tion follow with blindness, deafness, and spasticity, lead-ing to a state of decerebrate rigidity. These infants usu-ally die by 3 yr of age (2).

A change in the chemical composition of the brain ofsuch patients was first detected by Klenk who showedthat there was an increase in the ganglioside contentcompared with normal human brain tissue (3). Theparticular ganglioside which accumulated to the greatestextent was shown by Svennerholm (4) to be a minorcomponent of normal cerebral gangliosides, called Tay-Sachs ganglioside or ganglioside GM2, N-acetylgalac-tosaminyl (N-acetylneuraminosyl-) galactosyl glucosylceramide. The corresponding asialo-derivative of GM2called GA2 also accumulates (20% w: w compared withGM2) in the brain of Tay-Sachs patients. In the "O"-variant form of Tay-Sachs disease, the levels of GA2 inbrain are five times higher and the accumulation of GM2is somewhat more rapid than in patients with the classictype of this disease (5, 6). These patients also storedgloboside. Most recently, an "AB"-variant has been de-scribed which is clinically and chemically indistinguish-able from classical Tay-Sachs disease (called variant B)(7).

On the basis of previous information about the natureof the enzymatic defects in related lipid storage diseases(8), it is logical to look for a defect in the catabolism ofthe accumulated ganglioside and the corresponding asialocompound. Since the enzymatic degradation of GM2couldconceivably proceed either through the initial removal

The Journal of Clinical Investigation Volume 51 September 1972 2339

of N-acetylneuraminic acid to yield GA2 or N-acetylgalac-tosamine to yield N-acetylneuraminosylgalactosyl-glu-cosylceramide, a deficiency of either of the respective en-zymes might lead to an accumulation of the ganglioside.Assays for hexosaminidase with artificial substratesshowed that there was a higher total hexosaminidase ac-tivity in brain tissue from patients with Tay-Sachs dis-ease than that in controls (5). The possibility had to beconsidered that a neuraminidase deficiency was re-sponsible for the ganglioside accumulation. To examinethis alternative, GM2 was biosynthetically labeled in theN-acetylneuraminosyl portion of the molecule (9) andthe activity of this neuraminidase was determined. Thespecific activity of this enzyme was similar in tissuespecimens obtained from normal humans and patientswith Tay-Sachs disease (10). The question was thenpartially resolved by the demonstration that there weretwo hexosaminidase isozymes in normal human tissues( 11 ) and one of these, hexosaminidase A, was diminishedin tissue from patients with Tay-Sachs disease (12).Both isozymes are virtually absent in patients with the0-variant form of Tay-Sachs disease (13). In the AB-variant, the activity of each isozyme is increased whenchromogenic or fluorogenic substrates are utilized for theassay of hexosaminidase activity (7).

The activity of these hexosaminidases with regard totheir ability to catalyze the catabolism of the naturalsubstrate GM2has not been investigated thoroughly. Thisinformation is extremely important since widely diver-gent results have been obtained through the use of thechromogenic or fluorogenic substrates. The use of such"artificial" substrates does not necessarily imply a defectin the natural substrate cleavage. The catabolism of GM2labeled in the N-acetylneuraminosyl and N-acetylgalac-tosaminyl moieties had been reported in preparations ofskeletal muscle and the release of N-acetylgalactosarninewas diminished in such preparations from patients withTay-Sachs disease (10). However, the possibility of atwo-step catabolic scheme in which N-acetylneuraminicacid was first cleaved could not be ruled out using suchpreparations. We have resolved this uncertainty by thepresent investigations in which GM2 labeled either in theN-acetylneuraminosyl or the N-acetylgalactosaminylmoiety is used to study the catabolism occurring in nor-mal and Tay-Sachs tissue.

Additionally, because of the impending potential useof highly purified enzyme preparations in enzyme re-placement therapy in Tay-Sachs and other related dis-eases, we have studied the synergistic effect of addedpurified enzymes on the levels of hydrolyses in normaland Tay-Sachs lysosomal preparations.

METHODSMaterials. N-acetylmannosamine-[3H] (U) (SA 5.06

Ci/mmole) and uridinediphosphate N-acetylgalactosamine-

1-`4C (SA 43 mCi/mmole) were obtained from New Eng-land Nuclear Corp. (Boston, Mass.). Authentic GM2 wasprepared from frozen postmortem brain of Tay-Sachs pa-tients. 4-Methylumbelliferyl-p-N-acetyl-D-glucosamide waspurchased from Pierce Chemicals (Rockford, Ill.).

Radioactive GM2. N-acetylneuraminic acid-labeled GM2was prepared biosynthetically as previously described fromprecursor N-acetyl-D-mannosamine- [3H] (9). The specificradioactivity of the compound was 5.04 X 105 cpm/,umole.Hydrolysis indicated that radioactivity was associated onlywith the N-acetylneuraminic acid portion of the molecule.N-acetylgalactosamine-labeled GN12 was prepared from GM3and UDP-N-acetylgalactosamine-1-'4C as described (14).The specific radioactivity of this compound was 6.7 X 106cpmt//mole. Hydrolysis indicated that all of the radioactivitywas associated with N-acetylgalactosamine. Both com-pounds were chromatographically pure in three solventsystems.

For incubations with normal brain tissue preparations, theN-acetylgalactosamine-labeled GM2 was diluted to 1.0 X 106cpm/Amole. For Tay-Sachs brain preparations, this sub-strate was used without dilution because of the endogenousdilution (vide infra).

Preparation of cortical lysosomes. Human cortical graymatter was obtained at biopsy from "normal" and Tay-Sachs patients and was kept at 0° in Elliot's solution B(15) until use. The cortical gray matter was homogenizedby hand utilizing a TenBroeck glass homogenizer in 10 vol(w: v) of 0.25 M sucrose solution containing 1 mmEDTAat pH 7.4. Lysosomes were prepared by the procedure ofSellinger and Nordrum (16) with minor modifications.Evaluation of the activities of nine lysosomal marker en-zymes (including acid phosphatase, p-galactosidase, andsphingomyelinase) showed that the pellet obtained in thisprocedure was enriched three to fivefold over homogenatein the content of these hydrolases (17). This lysosomalpellet was resuspended in the original sucrose-EDTA solu-tion so that the final protein concentration was between 2and 3 mg/ml.

Experimental conditions. Utilizing the respective labeledganglioside, we have shown elsewhere' that the catabolismof GM2 can be initiated in similar lysosomal preparationseither by the sialidase reaction to yield reaction productswhich we have identified as N-acetylneuraminic acid andGA2 or by the hexosaminidase reaction to yield reactionproducts which were identified as N-acetylgalactosamine andGM3. Studies on the kinetic parameters of these reactionshave indicated that the optimal pH for the sialidase is 4.4in a citrate-phosphate buffer, that the reaction is propor-tional to protein content up to 400 ,ug protein per incuba-tion and with time up to 5 hr. Similarly, the optimal pHfor the hexosaminidase was 5.1 in a potassium acetatebuffer, and this reaction displayed similar proportionality toprotein and time. Since the effect of all detergents wasinhibitory, both of these reactions were carried out withoutadditional detergent. The procedure for the determinationof hexosaminidase activity utilizing the fluorogenic sub-strate has been described previously (17). Incubation con-ditions are summarized in Table I.

Correction for dilution with endogenous ganglioside. A0.2 ml portion of the resuspended lysosomes (400-600 jtgof protein) from control and Tay-Sachs patients was added

'Tallman, J., and R. 0. Brady. The catabolism of Tay-Sachs ganglioside in rat brain lysosome. J. Biol. Chem. Inpress. S

2340 J. F. Tallman, W. G. Johnson, and R. 0. Brady

TABLE I

Conditions of Incubation for Lysosomal Neuraminidase and Hexosaminidase Determinations

Concentration Concentration of Amount ofEnzyme Substrate of substrate buffer/pH protein Time

mM Ag hr

Neuraminidase AcneuGM2-3H 0.075 0.1 M Citrate-phosphate/4.4 200-300 4

GM2-hexosaminidase NAcgaIGM2-14C 0.100 0.1 M Potassium acetate/5.1 300-400 5

Total hexosaminidase 4MeUmb-Gluc NH2 0.25 0.15 M Citrate-phosphate/4.4 10-20 0.25

to 4 ml of a solution of chloroform-methanol, 2: 1 (v: v).The suspension was filtered and the residue on the filterpaper was washed with 2 ml of chloroform-methanol 1: 1.The filtrates were combined and the volume was reducedunder a stream of N2. The amount of N-acetylneuraminic

acid was determined on a portion of the residual solution(18) and the remainder was subjected to thin-layer chro-matography to isolate the individual gangliosides (19).More than 95% of the gangliosides in the lysosomal prepa-rations from the patients with Tay-Sachs disease was shown

Toy - Sachsgong I'los Ode

Standard Extractof Extract of StandardGM2 I ysosomes Mixed I ysosomes

Toy-Sochs gongliosides normalpatien t pat ient

FIGURE 1 Thin-layer chromatogram showing the accumulation of ganglioside GM2 inlysosomes of a patient with Tay-Sachs disease. The gangliosides became bluish purplewhen sprayed with resorcinol reagent (24). The two large slow-moving spots in thesecond and fourth lanes do not show this color reaction and are most likely someresidual sucrose from the density gradient centrifugation used for isolation of lyso-somes. Also present in the second and fourth lanes are various of the neutral lipidswhich show very high Rf. These also do not contain sialic acid.

Enzymatic Defect in Tay-Sachs Disease 2341

O 2.Al. ot q-N

GM3

TABLE I I

Gan-!iosile Catabolism by Human Brain L'.'sosa.o:cs

Enzyrm?

Hexosaminidase

Substrate4-methylumbellif- Sialidase

eryl-j6-GMs-14C N-acetyl-D- Gm23H

Source of tissue Age ganglioside glucosaminide ganglioside

pmoles/mg nmoles/mg pmoles/mgprotein/hr protein/hr protein/hr

Control seriesExploratory frontal lobotomy 21 yr 163 148 271Epileptigenic focus 25 yr 104 121 193Schilder's disease 7 yr 226 132 -*Abortus 13 wk 53 - -Lipidosis "unknown" - 107 - 187Degenerative disease 43 yr 186 216Accident victim or "trauma" 22 yr 143 - -

Mean 4SD 140±53 134411 217±33

Tay-Sachs patientsV. D. (classic) 28 months 0 513 225J. K. (classic) 20 months 0 1416 -

D. T. (variant) 13 months 6 3 232

Mfixed experimentDegenerative disease control - 138 - 210

and patient D.T. (theorv = 93) (theory = 224)

* not determined.

to be GM2 and appropriate corrections were made to correctfor the dilution of the labeled substrate. No GM2 was de-tected in the lysosomal preparations from the control brainspecimens (Fig. 1).

Preparation of supplemental enzymes. The details of thepurification of heart muscle neuraminidase (20), urinaryhexosaminidases A and B, and placental hexosaminidase A2will be published. Briefly, the neuraminidase is purified bygel filtration on Sephadex G-150 (Pharmacia, Uppsala,Sweden), chromatography on carboxymethyl Sephadex(CM-50) followed by isoelectric focusing. The purified en-zyme is enriched 3000-fold over the starting extract andthis enzyme catalyzes the hydrolysis of the molecule of N-acetylneuraminic acid from GM2. The urinary hexosamini-dase A was purified by ammonium sulfate precipitation,gel filtration on Sephadex G-200, DEAE-Sephadex, andcarboxymethyl Sephadex. The purified hexosaminidase is6000-fold purified over the initial activity. HexosaminidaseB from a similar protein extract was completely separatedfrom hexosaminidase A and purified 100-fold by ammoniumsulfate precipitation, Sephadex G-200, and DEAESephadex.Placental hexosaminidases were purified using much thesame techniques. The activity of these hexosaminidasesduring purification was assayed with the artificial substrate,4-methylumbelliferyl-p8-N-acetyl-D-glucosaminide.

2Johnson, W. G., and R. 0. Brady. Manuscripts in prepa-ration.

RESULTS

Lysosomes from fresh normal human brain contain en-zymes which catalyze the cleavage of both N-acetyl-galactosamine and N-acetylneuraminic acid moieties ofGm2 (Table II). The mean catalytic activity of GM2hexosaminidase in lysosomes obtained from the sevencontrol brain samples was 140 pmoles/mg of protein perhr. Approximately 62% of the total activity present incrude tissue could be recovered in this lysosome fraction.This enzyme was drastically diminished in similar prepa-rations from patients with Tay-Sachs disease in whichthe mean Gm2 hexosaminidase activity was 2 pmoles/mgof protein per hr. These values are corrected for dilutionby endogenous GM2. GM2 neuraminidase activity in thecontrol lysosomes averaged 217 pmoles/mg of proteinper hr and for two of the Tay-Sachs specimens, the meanwas 228 pmoles/mg of protein per hr. The recovery oftotal sialidase activity present in the crude tissue in thelysosomal pellet was 52% for control and 44% for Tay-Sachs. Mixing Tay-Sachs brain lysosomes with normalbrain lysosomes led to GM2hexosaminidase activity of 138pmoles/mg per hr and GM2neuraminidase activity of 210pmoles/mg per hr. The lysosomes obtained from the con-

2342 J. F. Tallman, W. G. Johnson, and R. 0. Brady

TABLE III

Edffect of the Addition of Purified Enzymes to Various Lysosomal Fractions

Addition 1 2 3 4 5 6 7 8 9 10 11

Neuraminidase - - - + - - - + + + +Urinary hexosaminidase A + - - + - - - + +Placental hexosaminidase A - + - - - - - - + +Urinary hexosaminidase B - - + + - - - + + + +Lysosomal extract*

Ovariant - - - - - + + - - aLysosomal extract1

Normal - - - - + - + 2-pmoles GaINAc

incubation/hr 0 14 0 38 49 1.2 63 23 0 21 0pmoles GaINAc/total mg

lysosomal protein/hr 186 6 138 180 0 164 0

* 2.4 mg protein/ml lysosomal extract. 80-,4l portions were used except where indicated by -.3.3 mg protein/ml lysosomal extract. 80-,.d portions were used except where indicated by 12.

trol brain specimens contained negligible quantities of GM2whereas similar preparations from the Tay-Sachs patientsshowed clear evidence of the accumulation of this gangli-oside (Fig. 1). In the lysosomes obtained from patientswith the classic form of Tay-Sachs disease, total hexosa-minidase activity measured with the artificial fluorogenicsubstrate was increased 4 and 10 times, respectively,over the mean of hexosaminidase activity in the con-trol preparations. As expected, total hexosaminidase ac-tivity in the brain lysosome preparation from the pa-tient with the O-variant form of Tay-Sachs disease con-tained only 2%o of that in the controls.

The effect of adding purified heart muscle neuramini-dase and human urinary and placental hexosaminidasesto the lysosome preparation obtained from the variantform of Tay-Sachs disease was examined (Table III).Neither of these purified hexosaminidase preparationssignificantly catalyed the hydrolysis of GM2 by them-selves in vitro. This result confirms the previous studiesof Sandhoff, Harzer, Wassle, and Jatzkewitz (7) andFrohwein and Gatt (21). However, when purified heartmuscle neuraiminidase was included in the incubation mix-ture with these enzymes, a small amount of N-acetyl-galactosamine was released from the lipid substrate.Conversely, the addition of purified hexosaminidase didnot enhance the GI2 neuraminidase reaction. In spite ofthe lack of Gm2 hexosamine-cleaving activity of purifiedhexosaminidases, the possibility existed that the addi-tion of these enzymes to lysosomes from brain tissue fromTay-Sachs patients might bring about the catabolism ofthe ganglioside either because of the presence in.lyso-somes of a necessary cofactor or because of an alteredsteric orientation of the Gr2 in situ. There was no evi-dence of release of N-acetvlgalactosamine-'4C when puri-fied hexosaminidases were added to the lysosomes from

the Tay-Sachs patient or when the very active sialidasewas added to such preparations.

DISCUSSION

Studies on the metabolism of gangliosides in brain ofpatients with Tay-Sachs disease were required for theexplicit identification of the enzymatic lesion in this con-dition. These investigations are hampered by the factthat brain tissue normally exhibits only very low ac-tivity with regard to the catabolism of the accumulatingganglioside (GM2). Steric hindrance of the terminal sugarhas been proposed as an explanation for this phenomenon.No one has yet been able to demonstrate the productionof either N-acetylgalactosamine or N-acetylneuraminicacid by colorimetric methods. Our radioactive productswere identified as the authentic sugar in each case andrepresented assay systems 500 times more sensitive thanthe colorimetric determination. The present experimentswere performed with GM2 specifically labeled with '4C inthe N-acetylgalactosaminyl moiety or in the N-acetyl-neuraminosyl portion with 3H in order to provide sen-sitive probes for delineating the pathway(s) of catabo-lism of this ganglioside. We have found that brain ly-sosomal preparations contain enzymes which catalyzethe cleavage of the N-acetylgalactosaminyl portion ofGin and the N-acetylneuraminosyl moiety by this method.The activity of the hexosamine-cleaving enzyme is vir-tually nonexistent in preparations of brain tissue ob-tained from patients with Tay-Sachs disease. The ac-tivity of the neuraminic acid hydrolytic enzyme in prepa-rations from Tay-Sachs patients was similar to that inthe control brain tissue specimens. Thus, the present ex-

periments extend and confirm the investigations withskeletal muscle biopsies that the enzymatic defect in Tay-Sachs disease studies is a deficiency of the hexosamine

Enzymatic Defect in Tay-Sachs Disease 2343

cleaving enzyme which normally is one of the pathwaysavailable for ganglioside catabolism in brain (10). Theother route via the neuraminidase is unaffected in Tay-Sachs disease. Although the values reported for theseenzymes are low, we feel that they reflect quite accuratelythe in situ activities for these enzymes. All catabolicschemes which have been proposed show GM2as an inter-mediate (22-24). This compound must be catabolized innormal tissue even at these low levels. It is significant tonote that these levels are sufficiently high to account forall GM2turnover (24).

In order to obtain a full understanding of the patho-logical biochemistry and physiology of Tay-Sachs dis-ease, the following observation must be also taken intoaccount. Hexosaminidase B which is present in thetissues of patients with the AB- and B-variant forms ofTay-Sachs disease can catalyze the hydrolysis of theasialo-GM2 produced via the neuraminidase pathway (7).Since GM2 neuraminidase is normal and the hexosamini-dase B isozyme is markedly increased in brain tissue ofthese patients and presumably able to function at leastinitially in Tay-Sachs patients, why does GM2accumulateat all? One reasonable explanation for this accumulationis the following. The activity of GM2 neuraminidase isquite low in brain preparations compared with that inother tissues (25). Since cerebral ganglioside turnoveris very rapid in the neonatal period of life, it may be as-sumed that a major portion of ganglioside catabolismmust also occur via the hexosaminidase pathway. Boththese enzymes catalyze rate-limiting reactions in gangli-oside metabolism.' Because the hexosaminidase is defi-cient in patients with Tay-Sachs disease, all GM2 catabo-lism must proceed exclusively via the neuraminidaseroute which, under these conditions, is insufficient toprevent the accumulation of GM2. Along with this accu-mulation, there are notable changes occurring in thelysosomal structure (17). The smaller amount of patho-logical involvement of systemic tissues (except for neu-rons in the myenteric plexus) in the classic form of Tay-Sachs disease my be due to the fact that the rapidity andquantity of ganglioside turnover in peripheral tissuesis much less than that in brain. Also, catabolism of GM2,peripherally, can also occur through the neuraminidasepathway and the combined activity of GM2neuraminidasewhich is functioning and hexosaminidase B which isaugmented in Tay-Sachs disease is sufficient to preventthe accumulation of a significant quantity of Tay-Sachsganglioside in non-neural tissues. The absence of evenhexosaminidase B in patients with the O-variant formof Tay-Sachs disease may be responsible for the evengreater accumulation of GM2 in the brain of these infantscompared with that in conventional cases of Tay-Sachsdisease (26).

A number of other observations which were made in

the course of these investigations deserve brief comment.The higher-than-expected rate of catabolism of GM2 bythe mixture of lysosomes from the normal and Tay-Sachsvariant may be related to the higher substrate concen-tration of GM2available for hydrolysis by the hexosamini-dase in the control lysosome preparation. Our choice ofGm2 concentration for use in the bulk of the assays per-formed (0.1 mM) was based on the known critical mi-cellar concentration of ganglioside. We wished to stayjust below this concentration in our experiments in orderto prevent aggregation of GM2. Thus we did not work atthe absolute V.a. of either the GM2 hexosaminidase orneuraminidase, although the reactions were carried outat optimal pH in a proportional range. Additionally, thecorrection applied to substrate specific radioactivity forthe presence of GM2in Tay-Sachs presumes that both theadded ganglioside and that in situ are in the same pool(or mix). The possibility exists that the stored Gr2 in theTay-Sachs lysosomes does not mix with the GM2-'4C andtherefore a low value is assumed for the radioactivity ofGM2 which is used to calculate the enzymic activity re-sulting in the higher value. Another possibility is thatthere is a cooperating factor in the Tay-Sachs lysosomeswhich when added to the hexosaminidase from normallysosomes increases its activity (27).

The inability of added purified enzymes to enhance sig-nificantly the enzymatic activities of the lysosomes maybe related to structural ordering of these enzymes withinthe lysosome. This postulated steric restriction mightalso contribute to the accumulation of GM2in Tay-Sachsdisease. The sialidase pathway may not be arranged inthe brain lysosomal membrane in an appropriate way todegrade the GM2 with facility. Furthermore, the pres-ence of an altered hexosaminidase A protein which binds,but cannot hydrolyze GM2, might prevent some of theGM2 from being catabolized by the sialidase. The patho-logical sequela of either of these constrictions is forma-tion of storage inclusion bodies and the deleteriouschanges in the neurons which are characteristic of Tay-Sachs disease. This finding also makes the possibilityof enzyme replacement therapy less appealing than mightotherwise be assumed. The inability of the enzymes towork in synergism will remain a problem in all attemptsat this therapy in Tay-Sachs patients.

A complete understanding of the catabolism of GM2de-mands a quantitative assessment of the relative contribu-tions of the neuraminidase and hexosaminidase pathways.The optimal pH for the neuraminidase in brain lysosomesis 4.4; the hexosaminidase has a pH optimum of 5.1.'The pH of the lysosomal environment is not known,and the possibility that the pH is not optimal forone or the other of these enzymes in normal or Tay-Sachs brain cannot be overlooked. We are at presentevaluating the contribution of each pathway under vary-ing conditions in control human brain preparations.

2344 J. F. Tallman, W. G. Johnson, and R. 0. Brady

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