Download C nuclear magnetic resonance studies of anaerobic

Bioscience Reports 3, 141-151 (1983)
Printed in Great Britain
141
13C nuclear magnetic r e s o n a n c e studies of
anaerobic
glycolysis in Trypanosoma b r u c e i spp.
N. E. MACKENZIE' 82 3ames Edwin HALL%,
I. W. FLYNNw and A. I. SCOTT*82
*Department of Chemistry, University of Edinburgh,
West Mains Road, Edinburgh EH9 3JJ, Scotland;
%Department of Parasitology and Laboratory Practices,
School of Public Health, University of North Carolina,
Chapel Hill, North Carolina 27514, U.S.A.; and
w
of Biochemistry, University of Edinburgh,
Edinburgh EH8 9XD, Scotland
(Received 24 January 1983)
A n a e r o b i c glycolysis in Trypanosoma brucei spp. has
been studied by 13C NMR at 50 and 75.5 MHz. The
uptake of [U-13C]glucose by cell suspensions of T. b.
b r u c e i was monitored by time-course spectroscopy, and
while no a n o m e r i c s p e c i f i c i t y was found, t h e . e n d products of glycolysis were confirmed as glycerol and
pyruvate together with alanine and dihydroxypropionate.
The i n t e r m e d i a c y of L-glycerol-3-phosphate was also
ascertained. The incorporation of C-I of [t-13C]glucose
and of C-6 of [6-13C]glucose into glycerol and pyruvate
in T. b. gambiense was quantified by measurement of
t h e l o n g i t u d i n a l r e l a x a t i o n t i m e s of the s p e c i e s
involved.
An incorporation to the extent of 66% of
each substrate into equimolar amounts of glycerol and
p y r u v a t e i n d i c a t e that Keq for the triosephosphateisomerase-mediated reaction approaches unity.
The use of high-resolution 13C nuclear magnetic resonance (NMR)
in the noninvasive monitoring of metabolism in vivo in both microo r g a n i s m s (1-3) and intact organs (4,5) is now well established.
These studies have typically involved the observation of chemical shift
c h a n g e s r e s u l t i n g from biochemical transformations of singly l~Cenriched substrates.
There still exists, however, the problem of the
rigorous assignment of carbon-13 nuclear-magnetic-resonance signals in
vivo and while u n a m b i g u o u s i d e n t i f i c a t i o n can only be made by
i s o l a t i o n of e n r i c h e d m e t a b o l i t e s this is not always possible or
practical. Comparison of extracts with authentic samples of suspected
compounds combined with variation of chemical shift with pH are most
commonly employed for the identification of unknown species.
82 Present address:
Center for Biological NMR, Department of
Chemistry, Texas A&M University, College Station, Texas 77843,
U.S.A.
01983
The Biochemical Society
142
MACKENZIE ET AL.
The use of multiply or uniformly laC-enriched substrates facilitates
the rigorous assignment of metabolites, especially transient specie%
and analysis of the more complex spectra obtained gives information
of cycling and pathway convergence (6) which may be obscured in the
case of singly 13C-enriched substrates.
In many studies involving singly enriched substrates much of the
d a t a e x t r a c t a b l e from the 13C NMR s p e c t r a o b t a i n e d from a
time-course has been used to draw conclusions about the kinetics of
t h e biological s y s t e m .
While the d a t a base is sound, certain
constraints of the technique should be fully realized before embarking
on c o m p l e x k i n e t i c arguments.
In particular the 13C-NMR signal
intensities obtained from spectra (even when normalized against an
i n e r t i n t e r n a l s t a n d a r d ) c a n n o t be used as a direct method of
quantification of metabolite concentration. Signal intensities are also
dependent on the longitudinal relaxation time (TI), which differs from
one carbon nucleus to another in a molecule; the extent of coupling to
adjacent and remote carbons; and the contribution from the nuclear
Overhauser e f f e c t .
Since these aspects have not been emphasized in
previous detailed analyses of spectra derived from metabolic processes
(2,7), we now report on IaC-NMR measurements of the anaerobic
metabolism of [U-laC]glucose by Trypanosoma b r u c e i b r u c e i and of
[l-13C]glucose and [6-13C]glucose by Trypanosoma brucei gambiense
which illustrate the above principles and, at the same tim% clarify
certain aspects of the glycolytic pathway examined in a previous study
of trypanosomes (8).
M a t e r i a l s and M e t h o d s
Tr ypanosomes
Both T. b. gambiense and T. b. brucei were used in these
experiments.
All handling procedures, including isolation and incubat i o n p r o c e d u r e s , for the monomorphic strain (TxTAT-I) of T. b.
g a m b i e n s e have been described (8).
The monomorphic strain (TREU
55) of T. b. b r u c e i used in these experiments was maintained as
stabilates at -70~
in 10% glycerol in Buffer B containing 50 mM
glucose.
Adult Wistar rats from a breeding colony at the University
of Edinburgh were inoculated intraperitoneally with--I06 trypanosomes.
B l o o d s t r e a m f o r m s were harvested by cardiac puncture of ethera n a e s t h e t i z e d rats at peak parasitemia approximately 72 h after
inoculation. Blood was collected into ice-cold 0.15% heparin in Buffer
B~ pH 8.0, containing 50 mM glucose. The blood was centrifuged at
1200 g for 10 min in an MSE 18 model centrifuge, the parasite layer
removed and washed once, in the above buffer, by centrifugation. The
trypanosomes were separated from remaining blood cells by column
chromatography on DEAE-cellulose (9) and washed once in cold Buffer
A or Buffer B, pH 7.4. The trypanosomes were resuspended to 5 x
108 cells/ml in Buffer A or B and kept on ice until incubation.
Incubat ion
Incubations were performed either in Buffer A or Buffer B to
which 13C-enriched glucose was added.
Immediately after incubation
t3C-NMR
STUDIES
OF
GLYCOLYSIS IN T. BRUCEI
1~3
an aliquot of trypanosome suspension was washed once by centrifugation with 4 vol. of ice-cold Buffer A or Buffer B, then resuspended to
5 x 108 cells/ml in ice-cold Buffer A or Buffer B.
Time-course experiments involved the addition of [U-13C]glucose to
15 ml of a cell suspension in Buffer A or Buffer B to give the final
c o n c e n t r a t i o n s d e s c r i b e d in e a c h f i g u r e .
The suspensions were
incubation at 35~
with shaking, under anaerobic (N 2) conditions.
Cell viability was monitored throughout by microscopic-examination.
Aliquots (2.5 ml) were taken at intervals (Table l) and immediately
frozen in an ethanol-dry ice mixture. These were allowed to thaw at
t~~ and were subjected to f r e e z e / t h a w cycles until cell lysis occurred.
13C-NMR spectra acquisition was performed on 2 ml of each aliquot
without further t r e a t m e n t after the addition of deuterium oxide (10%
v/v) as an internal lock. The remaining 0.5 ml of aliquot was used
for enzymatic assay.
Acid-lysed samples were prepared by addition of 6 M perchloric
acid, pelleting the precipitated protein by centrifugation at 1200 g for
l0 min, and r e a d j u s t i n g the supernatant to pH 7.4 with 0.33 M
potassium hydroxide.
Assays
The NMR s p e c t r a were recorded on either a Bruker WM 300
wide-bore spectrometer at 75.5 MHz or a Varian XL-200 spectrometer
at 50.5 MHz for 13C nuclei. All spectra were measured as indicated
in f i g u r e l e g e n d s , and ISC chemical shifts are given relative to
tetramethylsilane at 0 ppm.
Glycerol was assayed by a coupled enzymatic assay using glycerok i n a s e , p y r u v a t e kinase, and lactic dehydrogenase (10,11).
Since
pyruvate was present in all samples, the addition of glycerol kinase to
the reaction mixture was delayed until a stable absorbance at 340 nm
was obtained (12).
Pyruvate was measured by the standard method
(13) of sample incubation with NADH in the presence of lactate
dehydrogenase and measurement of the 'end-point' drop in absorbance
a t 340 nm b r o u g h t about by oxidation of the NADH.
Pyruvate
c o n c e n t r a t i o n s may then be calculated using the molar extincting
coefficient of 6.22 x 103 for NADH. Similarly, glucose was assayed
by addition of hexokinase.
Materials
Buffer A was the standard Roswell Park Memorial Institute (RPMI)
m e d i u m ( f o r m u l a t i o n 1640) p r e p a r e d by mixing the individual
chemicals (Sigma) according to the formulation but omitting glucose.
Buffer B was prepared by addition of sodium chloride (130 raM) and
potassium chloride (5 mM) to a 20 mM phosphate buffer at pH 7.4.
[ U - 1 3 C ] g l u c o s e (7996 atom enrichment) was purchased from KOR
Isotopes (Cambridge, MA). [l-I3C]glucose, [6-I3C]glucose (90% atom
enrichment), and deuterium oxide were purchased from Merck and Co.
(St. Louis, MO).
The glycerol and pyruvate assay materials were
purchased from Sigma.
144
MACKENZIE ET AL.
Table i. Anaerobic incubation 5 ~mol ml -I of
[U-13C]glucose by 5 x 10 8 cells ml -I of T. b. brucei
in Buffer A (see 'Materials and Methods')
Time
(min)
1
3
5
Ii
22
Concentrations
(~oI
ml -I) of
Motility
Pyruvate
G!ycerg~
Glucose
0.66
1.23
1.60
2.74
4.81
0.67
1.50
2.01
3.75
5.43
4.41
2.18
0.74
0.71
0.68
very low
R e s u l t s and D i s c u s s i o n
The bloodstream parasites T. b. brucei and T. b. gambiense are
the causative agents of nagana in cattle and sleeping sickness in man,
respectively.
The now generally accepted mechanism of glucose
catabolism in the bloodstream form of these eukaryotes is the
Embden-Meyerhof scheme, first proposed by Grant and Fulton (1$).
Under anaerobic conditions however, when equimolar quantities of
pyruvate and glycerol are produced (12,14), reservations have been
expressed as to the validity of this pathway (15,16).
Aerobically,
pyruvate is considered to be the exclusive end-product of glycolysis
(1%17) as it cannot be further metabolized, due to the absence of
both a functional tricarboxylic acid cycle (18,19) and the enzyme
lactate dehydrogenase (20) and is finally excreted in the host blood.
The transport of glucose, the sole energy source of bloodstream
trypanosomes, across the cell membrane has been determined as the
rate-limiting step in its catabolism (21).
Subsequent glycolysis is
extremely rapid and the steady-state concentrations of intermediates
so low as to v i t i a t e the monitoring of glycolysis by time-course
spectroscopy using [ I - 1 3 C ] - a n d [6-t3C]glucose (8).
This was c o n f i r m e d by incubation of [U-laC]glucose with cell
suspensions of T. b . b z u c e i . Although time-course spectroscopy failed
to detect any glycolytic intermediate, the gross features of anaerobic
metabolism could be observed.
The aJ~ equilibrium ratio of the
I a C - N M R signals of C - I of glucose is 36:64 (22) and in these
e x p e r i m e n t s does not change within experimental error throughout
glucose utilization.
This indicates that there is no increase in the
r a t e of a n o m e r i z a t i o n on the cell surface and that no anomeric
s p e c i f i c i t y exists in T . b . brucei, in contrast to other systems
(1,2,23).
Nine glycolytic enzymes and glycerol kinase are sequestered within
an organelle in T. b. b r u c e i and other trypanosome species.
The
existence of this glycosome has been invoked to explain the anaerobic
pathway converting glucose to equimolar pyruvate and glycerol while
giving a net yield of ATP (24).
Hammond and Bowman (25) have shown that glycerol formation
proceeds within the glyeosomal compartment of the trypanosome cell
via reversal of the glycerol kinase reaction resulting in ATP synthesis
by L-glycerol-3-phosphate(G-3-P)-dependent phosphorylation of ADP.
This t h e r m o d y n a m i c a l l y unfavorable reaction may proceed due to
I3C-NMR
STUDIES OF
GLYCOLYSIS IN T. BRUCEI
1#5
efficient compartmentation of the trypanosome cell leading to high
local concentrations of G-3-P.
This factor coupled with glucose
phosphorytation from G-3-P-dependent ATP formation would allow this
reaction to proceed in the direction of ATP generation (26).
The flux of [U-13C]glucose was conveniently monitored by freezing
aliquots of anaerobic incubation mixtures for assay at regular intervals
throughout the experiments, until microscopic examination revealed
that the ceils were no longer viable (Table I ) . This enabled t3C-NMR
data to be accumulated for much longer time blocks. The glycolytic
reaction profile in Buffer A obtained from the t3C-NMR spectra (Fig.
I A , B ) shows t h a t the only i n t e r m e d i a t e of glyco]ysis detected
anaerobically is G-3-P which is identilied by virtue of the 13C NMR
signal of C-3 (d, 668.I ppm; 3C2_C3 = 33 Hz).
This is the first
d i r e c t (i.e., n o n - e n z y m a t i c ) evidence for the existence of this
intraglycosomal metabolite in T. b. b r u c e 1 and confirms the dramatic
increase in the concentration of G-3-P found in cells undergoing
anaerobic glycosysis and in cells treated with salicilylhydroxamic acid
(SHAM), which mimics anaerobiosis (27).
The end-products of anaerobic glycolysis were shown to be glycerol
(Ct, 3 d, 663.~ 13Ct(3)_C2 = z~2 Hz; C 2 t, 673.4 13Cz_C1(3 ) = 42 Hz)
and pyruvate (C I dd, 6170.9 t3C1_C2 = 62 Hz, 23Ct_C3 = 13 Hz; C 2
dd, 6205.8 13C2_C1 = 62 Hz, 13C2_C3 = 30 Hz; C 3 dd, 627.3 13C3_C2 =
#0 Hz, 23C3_C1 = 13 Hz) (Fig. 2). The significant amount of alanine
formed anaerobically in Buffer A is due to transamination of pyruvate
(Table 1) enhanced by the amino-group donors present in this enriched
buffer.
These, however, must also be synthesized endogenously since
alanine (C I d, 6176.9 13C1_C9 = 60 Hz; C9 dd, 651.7 13C2_C! = 60 Hz,
13C2_C3 35 Hz; C 3 d 617.# ~3C3_Cz = 35"Hz) is produced in incubations in Buffer B in which no exogenous amino group donors are
present (Fig. 2). Alanine was found to be a consistent end-product of
glycolysis and although ignored in the past must be considered in any
future carbon-balance studies undertaken in this system.
In Fig. 1B
the apparently greater rate of alanine production compared with that
of p y r u v a t e simply r e f l e c t s the longer T t for C-3 of pyruvate
compared with T I for C-3 of alanine. The remaining 13C-NMR signals
in Fig. 2 are due to 2,2-dihydroxypropionate (C 1 d, 6179.8 3C1_C2 =
60 Hz; C2 dd, 695.2 13C2C3 = t~# Hz, t J c 2 _ c t : - 6 0 Hz; C~ d, 626.#
tJC3_C2 = ## Hz), arising from hydration of pyruvate (28-32), a
common reaction of a-oxoacids in aqueous media (33).
Despite the production of nearly equimolar quantities of pyruvate
and g l y c e r o l , as determined by enzymatic assay, there is a Iarge
discrepancy in the 13C-NMR resonance intensities of these metabolites.
This is most obvious on comparison of the resonances assigned to C1,3
of glycerol and C-3 of pyruvate (Fig. 2), an e f l e c t previously reported
(8) in the metabolism of [1-13C] - and [6-13C]glucose in suspensions
of T. b . g a m b i e n s e . It was found in the latter work that the label in
[ l - 1 3 C ] g l u c o s e was predominantly incorporated in CI,3 of glycerol
while C-6 of [6-13C]glucose was apparently randomized between CI,3
of glycerol and C-3 of p y r u v a t e .
This evidence could not be
rationalized in the light of the Embden-Meyerhof pathway and the
existence of an alternative mechanism was suggested.
In order to
reconcile this anomaly, [ l - t 3 C ] - and [6-13C]glucose were incubated
w i t h T . b . g a m b i e n s e essentially according to the method of
146
MACKENZIE
ET AL.
,
,,
4~
IQ-
2,
0
2
4
~
,
,0
,2
,4
,~
,,
,,~2
0
2
4
,
,
,2
,4
,~
20
22
TIMs
Fig. i. Normalized 13C-NMR signal intensities taken
from spectra (75.5 MHz) of 5 mM [U-13C]glucose (98%
atom enrichment) anaerobically metabolized by 5 x
108 cells/ml of T. b. brucei in Buffer A. A 10-mm
NMR tube was used and spectra were acquired at 5~
Peak intensities were taken from spectra which
r e s u l t e d from 5000 (72~
e90 = 25 s) pulses
accumulated over 38 min.
The lines were broadened
by 5.0 Hz by exponential multiplication of the
F.I.D.
The spectral width was 17 857 Hz of 16 K
data points. (A) O ~ signal intensity of e~8 C-6 of
glucose; A , signal intensity of C-1,3 of glycerol.
(B)
O , signal intensity of C-3 of L-glycerol
-3-phosphate,
A,
signal intensity of C-3 of
alanine; [] , signal intensity of C-3 of pyruvate.
Mackenzie et al. (g) but without addition of dithiothreitol to acid
lysates.
The spectra obtained were consistent with those published
previously (Fig. 3A,C) and the taC-NMR resonances assigned (see Fig.
2).
The l o n g i t u d i n a l r e l a x a t i o n time (T t) of the various species
involved were measured using the method of inversion recovery (34)
(Table 2).
While the T t values of ~C-1 and 8C-1 of glucose, C-3 of
a l a n i n e , and C-3 of 2 , 2 - d i h y d r o x y p r o p i o n a t e were of anticipated
magnitudes (35), C-3 of pyruvate was abnormally long at 10.68 +
1.27 s.
I~C-NMR STUDIES
OF
GLYCOLYSIS IN T. BRUCEI
1~7
G%X~
/',
-
o.,,, \
~
9'
It/.
p GI
\
Ckm~cal ~h~ft(pl~
Fig. 2.
13C-NMR (75.5 MHz) spectrum of the acid
lysate of an incubation involving the anaerobic
metabolism
of 5 m M [ U - 1 3 C ] g l u c o s e
(79% atom
enrichment) by 7.5 x 109 cells (5 x 108 cells/ml) of
T. b. brucei in Buffer B at pH 7.4 at 35~
A 20-mm
NMR tube was used and temperature was maintained by~
e m p l o y i n g a gated decoupling ratio of 5:1.
The
spectrum represents i0 200 (90~ ~90 = 40 s) pulses
accumulated over 6.26 h.
The lines were broadened
by 3.0 Bz by e x p o n e n t i a l multiplication of the
F.I.D.
The spectral width was 18 518 Hz over 16 K
data points. Abbreviations:
AI9 A29 A 3 - C-I 9 -29
and -3 of alanine 9 respectively; DI9 D29 D 3 - C-I
-29 and -3 of dihydroxypropionate9 respectively;
~98G19 egBG 6 - e- and 8-anomers of C-I and C-6 of
glucose 9 respectively; Gly I 3 and Gly 2 - C-I(3) and
.
9
C-2 of glycerol 9 respectlveIy; PI~ P2~ P3 - C-19 -2~
and -3 of pyruvate, respectively.
Table 2. Longitudinal relaxation times (T I)
measured by the method of inversion recovery
Carbon-13 @pecies
T 1 (s)
eC-i Glucose
0.57 +- 0.06
8C-I Glucose
0.81 + 0.05
C-193 Glycerol
1.34 + 0.07
C-3 Dihydroxypropionate
1.17 +_ 0.34
C-3 Alanine
2.81 + 1.49
C-3 Pyruvate
10.68 -+ 1.27
Its8
MACKENZIE
A
ET
AL.
B
fpr~
~,'GlYl,3
J~
I
I
I
I
1
I
IO0
I
1
f
~1
I
0 I00
C
.... I
0
D
/~,pG6
I
IL]O
I
I
I
I
I
PPM
0
I
IL]O
I
I
I
I
PPM
Fig. 3.
(A) 13C-NMR (50.3 MHz) spectrum of an acid
lysate of 5 m M [l-13C]~glucose (90% atom enrichment)
anaerobically metabolized by 5 x 108 cells/ml of T.
b. 9ambiense in Buffer B. A 10-r~n NMR tube was used
and the spectrum was acquired at 0~
The spectrum
represents
7200 (50~
~90 = 14.2 ps) pulses
accumulated over I h.
The lines were broadened by
0.45 Hz by exponential multiplication of the F.I.D.
Abbreviations same as for Fig. 2.
(B)
13C-NMR (50.3 MHz) spectrum of the same sample
as in (A) above acquired at 0~
with no nuclear
O v e r h a u s e r enhancement.
The spectrum represents
I
0
I3C-NMR
STUDIES OF
GLYCOLYSIS IN T. BRUCEI
1#9
S p e c t r a of the acid l y s a t e of the cell suspensions were then
a c c u m u l a t e d under c o n d i t i o n s of z e r o nuclear Overhauser e f f e c t
(decoupler power o f f for 10 s~ on for 0.5 s during F.I.D.) and a small
flip angle (29 ~ ) to minimize differential saturation e f f e c t s (36).
Under these conditions, which optimize the C-3 of pyruvate signal/
noise~ integration of the t3C-NMR resonances gave a true representation of the relative intensities if the integral for the C-3 of pyruvate
was corrected by +g%.
The resonances in Fig. 3B were integrated,
adjusted as above, and it was found that C - 1 of [ l - t 3 C ] g l u c o s e is
incorporated preferentially into C-1 (= C-3) of glycerol to the extent
of 2 to 1 over C-3 of pyruvate, alanine, and 2,2-dihydroxypropionate
c o l l e c t i v e l y (the l a t t e r t w o m e t a b o l i t e s arising from pyruvate).
I n t e g r a t i o n of the r e s o n a n c e s in Fig. 3D revealed that C-6 of
[6-t3C]glucose was preferentially incorporated in the same ratio (2/1)
i n t o C - 3 of p y r u v a t e ,
alanine,
and 2 , 2 - d i h y d r o x y p r o p i o n a t e
(collectively) over C-1~3 of glycerol.
Triosephosphate isomerase interconverls glyceraldehyde-3-phosphate
( G a l d - 3 - P ) , d e r i v e d from C-6 of g l u c o s e , and dihydroxyacetone
phosphate (DHAP), the initial site of C-I of glucose. The equilibrium
constant (Keq) has been assigned the value 22 to 2# (37,3g) for
DHAP/Gald-3-P in rabbit-muscle enzyme.
More recently, however~ it
was determined that in neutral solutions, Gald-3-P is 96.7% hydrated
to the gem diol and DHAP is ##% hydrated (39,#0). Since only the
unhydrated substrates are bound by the enzyme, the corrected value of
his 367 (DHAP/Gald-3-P) at 25~
Ke lc~ trypanosome glycolysis an earlier study (1#) showed that under
a n a e r o b i c c o n d i t i o n s C-1 of [1-t~C]glucose was preferentially incorporated into glycerol despite equimolar production of this metabolite and pyruvate as end-products. It was concluded from this result
that the rates of formation of glycerol from DHAP and pyruvate from
Gald-3-P were equal and greater than the rate of the triosephosphate-isomerase-mediated conversion of Gald-3-P into DHAP.
We have shown in this study that C-1 of glucose is incorporated
into glycerol to the extent of 66%, in agreement with earlier results
(1#). However, the same incorporation (66%) of C-6 of glucose into
p y r u v a t e and the e q u i m o l a r production of glycerol and pyruvate
i n d i c a t e s that Ke
for t h e t r i o s e p h o s p h a t e i s o m e r a s e r e a c t i o n
approaches unity in ~. b r u c e i spp.
4000 (29 ~ ~90 = 14.2 ~s) pulses with an acquisition
time of 0.5 s and delay time of 10 s. Lines were
broadened as above by 1Hz.
(C)
13C-NMR (50.3 MHz) spectrum of an acid lysate
of 5 m M [6-13C] glucose (90% atom enrichment)
anaerobically metabolized by 5 x 108 cells/ml of T.
b. gambiense in Buffer B.
Spectrum accumulation
condition as for (A) above.
(D)
13C-NMR (50.3 MHz) spectrum of the same sample
as in (C) above accumulated under conditions as for
(B) above.
150
MACKENZIE
ET AL.
R a t i o n a l i z a t i o n of this incongruity c a n n o t be found in the high
degree
of c o m p a r t m e n t a t i o n in T. b r u c e i spp. as no t r a n s m e m b r a n e
processes are involved.
This is d u e t o t h e i n h i b i t i o n of the
p r o m i t o c h o n d r i a l G-3-P oxidase under a n a e r o b i c conditions which causes
the concentration
of c y t o s o t i c DHAP t o rapidly diminish and so
i n a c t i v a t e the p u t a t i v e s p e c i f i c t r a n s l o c a t o r for DHAP in the DHAPG-3-P s h u t t l e (41).
Acknowledgements
We wish to thank the S c i e n c e and Engineering R e s e a r c h Council for
support and Miss Sandra Gilchrist for t r y p a n o s o m e p r e p a r a t i o n s .
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