Induction of nucleoside transport sites into the host cell membrane of Babesia bovis infected erythrocytes

Molecular and Biochemical Parasitology, 35 (1989) 269-276 Elsevier

269

MBP 01172

Induction of nucleoside transport sites into the host cell membrane of Babesia bovis infected erythrocytes A n n e t t e M. G e r o School of Biochemistry, University of New South Wales, Kensington, N.S.W., Australia (Received 24 November 1988; accepted 14 February 1989)

Normal bovine erythrocytes have negligible ability to transport adenosine and related nucleosides across their cell membrane. However, infection with the intraerythrocytic parasite Babesia bovis was found to induce a nucleoside permeation site into the host cell membrane. Transport experiments over periods of up to 30 s determined that the transport rate of 1 0,M adenosine into the infected cell was 1.72 --- 1.2 pmol incorporated (Ixl cell water)-ls -1, a rate three times higher than for normal human erythrocytes. Incorporation studies over 6 h with labelled adenosine indicated that the purine moiety was incorporated into parasite nucleic acids. The mammalian nucleoside transport inhibitors, nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole inhibited the induced nucleoside transport mechanism in the Babesia-infected erythrocytes, though at higher concentrations than those required to inhibit normal human erythrocyte transport. An IDs0 value for NBMPR of 0.36 txM was determined. Phloretin and 5'-p-fluorosulphonyl benzoyl adenosine-HCl (5FSBA) were also shown to be inhibitory, with IDs0 values of 0.11 and 0.18 I~M, respectively, whilst phlorizin and verapamil at 1 IxM had no effect. Binding studies with [3H]NBMPR indicated that high-affinity NBMPR binding sites could not be detected in either normal or B. boris infected bovine erythrocytes. The results indicate that the induced nucleoside permeation site(s) in B. bovis infected erythrocytes has characteristics different from either human erythrocytes or erythrocytes infected with the malarial parasites Plasmodium falciparum or Plasmodium yoelii. Key words: Babesia bovis; Plasmodium falciparum; Nucleoside transport; Nitrobenzylthioinosine; Adenosine; Altered nucleoside permeation; Bovine erythrocyte

Introduction

Many similarities exist between the two diseases malaria and babesiosis, in their clinical manifestations, their intraerythrocytic development in the mammalian host and their antigenic properties [1,2]. Notably, the intraerythrocytic parasite of cattle, Babesia bovis, causes a marked change in the properties of the infected erythroCorrespondence address: Annette M. Gero, School of Biochemistry, University of New South Wales, Kensington, N.S.W., 2033, Australia Abbreviations: NBMPR (nitrobenzylthioinosine), 6-(4-nitrobenzyl)thio]-9-13-D-ribofuranosylpurine; NBTGR(nitrobenzylthioguanosine), 2-amino-6-[(4-nitrobenzyl)thio]-9-13-D-ribofuranosylpurine; phlorizin (phloretin-2-13-D-glucoside); 5FSBA, 5'-p-fluorosulphonyl benzoyl adenosine-HC1; PBS, phosphate-buffered saline, pH 7.4.

cyte [3] and an alteration of the host cell erythrocyte membrane analogous to the changes induced in the host membrane by the human malarial parasite Plasmodium falciparum [4]. Many of the changes in cells infected by either P. falciparum or Babesia species are related to the necessity for the intraerythrocytic parasite to obtain compounds for its own metabolic requirement which are not synthesised by the host cell, an important example being the salvage of purine nucleotide precursors [5-11]. Neither B. bovis nor P. falciparum has the ability to synthesize de novo the purine ring; they are therefore dependent on the salvage of preformed purines from the host [9]. We have determined a marked change in the permeation characteristics of adenosine and the cytotoxic adenosine analogue, tubercidin, following infection of human erythrocytes by P. falciparum

0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

270 [10,11]. The mammalian nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine (NBTGR), dipyridamole and dilazep were ineffective, at concentrations several orders of magnitude higher than required to block transport in normal erythrocytes, at blocking an apparently induced component of the transport of these nucleosides in infected cells. The ineffectiveness of the nucleoside transport inhibitors was related to the stage of development of the intraerythrocytic parasite and insensitivity was greatest at the trophozoite or schizont stage for both chloroquine-sensitive and chloroquine-resistant strains. Binding studies of [3H]NBMPR to both normal erythrocytes and those harbouring P. falciparum parasites at each morphological stage, indicated that fewer highaffinity NBMPR binding sites were present on cells containing mature parasites than on the uninfected cells, although the permeation rate was the same or greater than in normal erythrocytes [10]. The changes during parasite maturation to the trophozoite or schizont stage, suggested the presence of an induced nucleoside permeation site relatively insensitive to NBMPR. Normal bovine erythrocytes, unlike human red cells, do not appear to transport nucleosides [12]. In this paper, evidence is provided that the presence of the Babesia parasite in the bovine erythrocyte induces nucleoside permeation sites into the host cell membrane. A preliminary comparison of its characteristics with the human erythrocyte nucleoside transporter and with the altered nucleoside permeation site following infection with P. falciparum is described. Materials and Methods

Materials [G-3H]NBMPR (23 Ci mmol 1), was purchased from Moravek Biochemicals, Brea, CA, U.S.A., and purified by Dr. A.R.P. Paterson, Edmonton, by high-performance liquid chromatography using a C18 reverse phase column eluted with methanol/water gradients and stored in the same solutions at -20°C. [2,5',8-3H]Adenosine (40 Ci mmol-1), [6-3H]thymidine (23 Ci mmol-l), [5-3H]uridine (28 Ci mmol-l), [U-X4C]sucrose (540 mCi mmol 1) and [3H]H20 (5 mCi mmo1-1) were

obtained from Amersham International, Amersham, U.K. Unlabelled NBMPR and NBTGR were obtained from Sigma, St. Louis, MO, U.S.A. Saturated solutions of NBMPR and NBTGR (about 45 and 57 txM, respectively) were obtained by stirring in phosphate-buffered saline, pH 7.4 (PBS) for 2 h at room temperature, as described previously [11]. Dipyridamole, verapamil, phloretin, phlorizin and 5'-p-fluorosulphonylbenzoyl adenosine HC1 (5FSBA) were purchased from Sigma. Dilazep was a gift from Dr. J. Wiley, Austin Hospital, Heidelberg, Victoria, Australia. 2,5-Diphenyloxazole (PPO) was obtained from Packard Instruments International, Zurich, Switzerland. Silicone oils were obtained from Dow Corning Australia, Blacktown, Australia. A silicone oil mixture of density 1.03 g 1-1 was prepared by mixing (part by weight) oils of three grades: oil 702 (45 cSt viscosity, 80 parts), oil 200 (1.5 cSt viscosity, 14 parts), and oil 200 (5 cSt viscosity, 7 parts). Methods Babesia bovis. Virulent B. bovis (Samford strain) [13] was passaged through two splenectomised calves and blood was collected on the day of experimentation into citrate phosphate dextrose [14]. White cells were removed by passing the blood through a column of cellulose powder (Whatman C F l l ) [15]. Suspensions of 70-100% infected red blood cells were prepared b3~ differential lysis in hypotonic saline [3]. Packed cells were suspended in 5 volumes of 0.45% NaC1 for 5 s followed by centrifugation at 3000 × g for 10 min to produce a pellet which contained 70-100% infected erythrocytes. The pellet was washed twice in PBS, pH 7.4, and suspended at a volume of 2 x 108 cells m1-1 for the use in transport assays. Human erythrocytes and uninfected control bovine blood samples were obtained on the day of experimentation and were treated in a similar manner, with the exclusion of the hypotonic saline lysis step. Nucleoside transport measurements. Nucleoside transport over short time intervals was measured by an established procedure [10]. Cells (2 × 108 m1-1) in 100 Ixl PBS were added to Eppendorf centrifuged tubes containing 150 t~1 silicone oil

271

mixture (1.03 gl -t) with 100 Izl of 3H-labelled permeant layered on top of the oil. Transport intervals (1-30 s at 25°C) were ended by centrifugation at 16 000 x g for 30 s which pelleted cells under the oil layer. The density of the oil layer was adjusted so that separation of the unused permeant layer and the cell sample was obtained by centfifugation. It was also established that free merozoites, leukocytes and infected erythrocytes which were broken or lysed did not enter the oil layer during centrifugation. Thus, the radioactivity incorporated into the cell pellet below the oil represented only the fraction transported across the membrane of an intact normal or infected erythrocyte. Transport of nucleosides was determined by this method over time intervals as short as 3 s. Control experiments with human erythrocytes stopped with 100 Ixl 20 IxM NBMPR and compared with the oil stop method indicated that the separation of the cells from the radioactive permeant, following the initiation of centrifugation through the oil layer, took 2 s. Background radioactivity due to the extracellular volume of permeant which was present in the cell pellet was measured by control experiments containing [Ut a c ] - s u c r o s e in the medium in place of the nucleoside permeant. 3 H 2 0 w a s used to determine total cell water space. The cell pellet was processed as described previously [16] and counted in a Packard Model 300C liquid scintillation spectrometer.

Equilibrium binding of [3H]NBMPR. Binding of NBMPR to high affinity sites on normal bovine erythrocytes and infected cells was measured under equilibrium conditions as described by Gati et al. [17]. Cells (2 x 107 m1-1) were incubated for 30 min at room temperature in 1-ml portions of PBS containing graded concentrations of [3H]NBMPR (0.125 nM-10 nM). Non-specific binding of the ligand was determined in separate samples that contained, in addition, 20 ~M NBTGR, a congener of NBMPR that binds tightly at the high-affinity inhibitor site. After incubation, the cells were pelleted by centrifugation at 16000 x g for 1 min. Portions of the supernatant were assayed for 3H to determine equilibrium free concentrations of the unbound ligand. The pellets were washed once with 1 ml ice-cold PBS.

After suspension in 30 txl PBS, pellets were extracted with 1 ml ice-cold alcohol mixture (90% ethanol, 5% methanol, 5% isopropanol) for 30 min, a procedure that quantitatively solubilised site-bound [3H]NBMPR. The extracts were clarified by centrifugation for 2 min at 16 000 x g and 3H w a s measured in the resulting supernatants. Dissociation constant (Kd values) for the NBMPR-binding site complex and binding site densities (Bma x values) were determined by computer-assisted analysis of binding data [18]. Results

Transport studies. The permeation of [3H]adenosine (1 txM) could not be detected in normal bovine erythrocytes (Fig la) in either transport experiments from 0-10 s, or over a 1-h period. In each experiment, human erythrocytes were included as a positive control (Fig. lb); in these cells adenosine transport over a period of 10 s was totally inhibited by 1 IxM NBMPR. By contrast, erythrocytes infected with B. bovis, which had been concentrated by hypotonic lysis from a mixture of infected and normal cells, were shown to transport adenosine at a greater rate than human erythrocytes but were not inhibited

2b

121° "8

(3.

k

rl Bovinerbc

I

# B 4- NBI,4PR

l

I 0

I 2

I I I 4 6 8 Time (see)

I 10 12

,/

L

I'2 Time

(see)

Fig. 1. Transport of adenosine into bovine (panel a) and human erythrocytes (panel b). The transport of 1 IxM [all]adenosine into normal red cells (A) and bovine erythrocytes (o) was measured by exposure of the cells to the permeant for short periods followed by centrifugation of the cells through an inert oil layer. Transport in the presence of permeant + 1 tLM NBMPR (A ,0) was measured by the addition of ceils to a mixture of adenosine and NBMPR. The total radiolabel incorporated into the cell pellet was calculated by subtraction of the background attributable to the radiolabel in the extracellular water space in the pellet. Time was calculated from the initiation of centrifugation plus 2 s for centrifugation through the inert oil layer.

272

~_

~.

75°/° babesia

~8 6

75"1° + NBMPR (1 v M )

T1. < :Z

A 4

1" .&/

.--"" A

./r" ~ Human rbc N

2

o_ 0

14*/o babesia

....

"....

~- ......

14*/, 4- NBMPR (I~JM) 8 o v i n e rbc H 4-NBMPR (1/JM)

Time (see)

Fig. 2. A comparison of [3H]adenosine transport in normal bovine (~), human (×), and Babesia-infected (&,u) erythrocytes in the presence and absence of 1 ixM NBMPR over periods of up to 12 s. Each curve represents the results of two or more experiments, carried out as described for Fig. 1. totally by 1 IxM N B M P R (Fig. 2). Normal bovine cells were also exposed to serum from highly infected cows for periods of up to 1 h, washed twice in PBS and assayed for adenosine transport. The permeation of adenosine was still negligible in these cells, indicating that the induction of adenosine transport in the infected bovine ceils was not due to a component in the serum from infected animals. The amount of adenosine transported into Babesia-infected cells was found to be proportional to the percentage of infected cells present in each preparation, irrespective of whether the cells had been subjected to the lysis step or not. As the influx of adenosine in the concentrated infected cells was linear up to at least 10 s, (Fig. 2), the slope represented the initial rates of permeant transport and hence nucleoside transport rates. From the data for the transport rate of a preparation of 75% infected cells, it was calculated that a preparation of 100% Babesia-infected cells would transport 1.72 +- 1.2 pmol adenosine (Ixl cell water)-Xs -1 (approx. 3 times more than for normal human erythrocytes.). Calculations of the concentration inside and outside the m e m b r a n e indicated that adenosine equilibrated in less than 3 s. However, the intracellular concentration continued to increase over that present in the medium, indicating that the conversion of adenosine to metabolites occurred

within the infected erythrocyte. Preliminary data, which investigated the concentration dependence of adenosine influx, suggested that the Babesia permeation may occur via a mediated nucleoside pathway with, however, a very high K m for adenosine of 1.8 mM (data not shown). The incorporation of [3H]adenosine (30.6 pmol (txl cell water) -1 over 6 h) into T C A precipitable material provided evidence that the Babesia-infected erythrocyte had the capability to incorporate adenosine into R N A and DNA. Similar studies were carried out with the pyrimidine nucleosides thymidine and uridine (1-100 ~M) for periods of 0-30s. Both nucleosides were transported significantly less than adenosine, with permeation rates for thymidine and uridine of 0.21 + 0.1 pmol (~1 cell water)-ls -1 and 0.09 -+ 0.03 pmol (Ixl cell water) ~s-1, respectively. However, both thymidine (25 txM) and inosine (15 I~M) were shown to inhibit the permeation of 1 IxM adenosine by approx. 50%. Inhibitor studies. Non-physiological inhibitors of mammalian nucleoside transport were also tested over 4 s for their activity against adenosine transport by the Babesia 'transporter'. The results are shown in Table I and compared to those for human erythrocytes. NBMPR, N B T G R , dilazep and dipyridamole showed inhibition at 1 IxM of approx. 80-90%, for the induced Babesia nucleoside permeation system. Phloretin, a modifier of the membrane dipole potential, was also an excellent inhibitor at 1 ~M. However, phlorizin (phloretin-2-[3-D-glucoside), a compound which has been found to inhibit transport through pores in erythrocytes infected with the malarial parasite, P. falciparum [19], and verapamil, a Ca 2+ channel blocker in human erythrocytes [20,21], had no effect. The adenosine analogue 5FSBA also inhibited about 90% of the adenosine permeation at 1 p~M. Neither phloretin (1 pxM) nor 5 FSBA (1 p~M) showed any inhibitory effects on the transport of adenosine into human erythrocytes, whilst the established mammalian nucleoside inhibitors N B M P R , N B T G R , dilazep and dipyridamole caused complete inhibition of adenosine transport at the concentration used (Table

I). The effects of increasing concentrations of

273 TABLE I Inhibition of adenosine transport

Babesia-infected Human erythrocytes erythrocytes (% inhibition) (% inhibition)

Compound

NBMPR

1 p,M 10 ~M

80.0 97.0

98 99

8C

_=*

6c

E

NBTGR

1 ixM 10 ~M

92.7 96.3

98 99

Dipyridamole 1 ixM 10 ~M

95.5 96.8

98 99

Dilazep

1 IzM 10 p,M

78.0 96.2

98 99

Phloretin

1 IzM 10 ~M

94.0 95.7

3 nd

5FSBA

1 IzM 10 IzM

88.7 nd

3 37

Phlorizin

1 v.M 10 v.M

0 32

0 12

Verapamil

10 IzM

0

0

Human

NBMPR on adenosine influx rates in preparations of 97% Babesia-infected erythrocytes, compared to human erythrocytes, are shown in Fig. 3. The IDs0 value of NBMPR in human erythrocytes was calculated as 20 nM, which may be compared to the value previously reported as <30 nM [17]. By comparison, the ID50 for NBMPR in Babesia-infected cells was 0.36 IxM, a value approx. 10 times higher than that reported for human erythrocytes, but some 50 times lower than the value (20 ~M) we obtained for the rodent malaria parasite Plasmodium yoelii [17]. Assuming that the non-specific binding of NBMPR to each cell type was similar, and noting that the number of cells in each assay was similar in each respective assay (2 × 108 cell ml-]), these results may demonstrate significant differences between the NBMPR sensitivity in the three types of nucleoside 'transporters' . ID50 values for Babesia-

RBC

~ Babesia

~ 2o ~6 -14

Inhibition of influx of 1 I~M adenosine over a period of 4 s into a preparation of 97% Babesia-infected cells by established inhibitors of mammalian nucleoside transport compared to identical experiments using human erythrocytes, nd = not determined.

a

-~1

'

,

_~

,

,

...~

,

L

-2

Log I-.,~MP.-j M Fig. 3. Effect of graded concentrations of NBMPR on adenosine transport into human erythrocytes (n) and a preparation of 97% Babesia-infected erythrocytes (e). Transport of adenosine in the presence of NBMPR (10 -4 - 10-]2M) was carried out as described for Fig. 1. after a 4-s period of exposure of the cells to a mixture of the permeant and NBMPR. Each curve is the result of two or more experiments.

infected erythrocytes were determined for phloretin and 5FSBA as 0.11 IxM and 0.18 ~xM, respectively.

[3H]NBMPR-binding studies. To investigate the differences between the normal bovine erythrocyte and the erythrocyte infected with B. boris, the high-affinity NBMPR binding site content of each cell type was measured. It was of particular interest to determine whether the induced nucleoside permeation site in the membranes, of the infected cells also contained induced NBMPR binding sites. As high-affinity NBMPR sites could not be detected for normal bovine erythrocytes, normal human erythrocytes were used as a positive control. A Scatchard analysis of data for sitespecific high-affinity equilibrium binding of [3H]NBMPR on human erythrocytes indicated the presence of a single population of high-affinity sites with a Bma x value of 9900 molecules cell -1 and a K d = 0.1 nM, which is in good agreement with previously published values [22]. However, no high affinity equilibrium NBMPR binding of sites of similar affinity could be detected on the erythrocytes infected with B. bovis.

274 Discussion

Nucleoside transport in free-living parasites investigated to date, including Leishmania donovani promastigotes [23], Schistosoma mansoni or Schistosoma japonicum [24,25], Trypanosoma gambiense [26] and Trichomonas vaginalis [27] appears to occur via sites which are relatively insensitive to micromolar concentrations of the established mammalian nucleoside transport inhibitors. It was of considerable interest, therefore, to determine the influence of the intraerythrocytic Babesia parasite on the nucleoside permeation of the host bovine cell membrane. We have previously found that intraerythrocytic P. falciparum [10] and P. yoelii [17] appear to induce a new transport component into the host membrane, which is relatively insensitive to micromolar concentrations of inhibitors of mammalian nucleoside transport. The advantage of using the bovine Babesia system to examine the transport of nucleosides in the Babesia-infected erythrocyte has been that no host cell transporters are present in the uninfected bovine erythrocytes, so that it is possible to only have to consider the characteristics of an induced transporter. The induced Babesia nucleoside permeation site was shown to incorporate the purine nucleoside, adenosine, into the infected cell at a rate 3 times higher than for the human erythrocyte. However, the pyrimidine nucleosides thymidine and uridine, appeared to be transported only very slowly. By contrast, uridine is transported efficiently in human erythrocytes [28]. Although adenosine influx was found to show saturating behaviour, the high K m obtained and the inhibition by relatively high concentrations of thymidine and inosine do not necessarily indicate that the induced permeation site is a transporter protein. There is some conjecture in the literature regarding the characteristics of the permeation mechanism of small molecules in the Plasmodium-infected erythrocyte [4,29]. Development of the malarial parasite inside the host cell erythrocyte induces changes in the permeability of the cell to glucose [30], anions [31], amino acids and polyols [32-35], as well as nucleosides [10,11,17]. These changes may be due to the insertion of

parasite-specific proteins into the host cell membrane, to form loose seals with the hydrocarbon chain of the membrane phospholipids, which results in leaks or membrane pores [29]. It has also been suggested that either existing host cell transporters may be modified by the intracellular parasite, or that parasite encoded transporters may be inserted into the host cell membrane. At this stage it has not been possible to discriminate between these possibilities in the Babesia-infected cells. However, further experiments (A.M.G. and I.G. Wright, unpublished data) have demonstrated that polyclonal antisera from Babesia-infected animals are able to inhibit adenosine transport into Babesia infected erythrocytes, suggesting that the permeation site represents an alteration of the host cell membrane which is recognisable by the immune system. The properties of the Babesia-induced 'transporter' have similarities and differences to the induced transporter in Plasmodium spp. [10,17]. In P. falciparum, the induced nucleoside transporter appears to be insensitive to NBMPR up to at least 10 p~M, and similarly insensitive in P. yoelii-infected cells. By contrast, the Babesia-induced 'transporter' was inhibited by NBMPR with an IDs0 value of 0.36 p~M, and the inhibition by other mammalian transport inhibitors NBTGR, dipyridamole and dilazep, was similar. The absence of high-affinity NBMPR binding sites further illustrates the differences between the parasite-induced and the normal erythrocyte nucleoside transporters. In human red blood cells NBMPR binding sites appear to be associated with a functional nucleoside transporter. However, it appears that the NBMPR binding site may not always be an integral part of the nucleoside transport protein, as in some cell lines, for example Walker 256 rat carcinosarcoma cells, a transport system of low NBMPR sensitivity is present and NBMPR binding sites are absent [36,37]. In $49 mouse lymphoma cells, NBMPR binding and nucleoside transport are genetically distinct [38]. Thus there is the possibility that the parasite-induced permeation site may have a different relationship to NBMPR than the human host erythrocyte. If, as suggested by Sherman [4], the induced transport process in the malaria-infected erythrocytes may be common to several

275

metabolites (such as nucleosides, glucose or amino acids), then transport inhibitors such as verapamil, phlorizin and phloretin might be expected to affect the permeation of nucleosides. It was found that nucleoside transport in Babesia-infected cells was not inhibited by verapamil and phlorizin at 1 ixM but was, however, inhibited by phloretin (IDs0 = 0.11 IxM) and 5FSBA (IDs0 = 0.18 txM). Phlorizin has been reported to interfere with transport pores in P. falciparum-infected cells [19] and phloretin, a surface dipole modifier which affects many carrier-mediated as well as non-mediated transport systems [39], has been demonstrated to block glucose-induced haemolysis in P. falciparum-infected cells [40]. The observation that phloretin and 5FSBA are inhibitors of nucleoside transport in Babesia-infected cells is worthy of further investigation, particularly as they appeared to have no apparent inhibitory activity against nucleoside transport in normal human erythrocytes. The induction of the 'transporter' in Babesiainfected cells also provides a unique opportunity for the possible application of chemotherapy in directing toxic nucleosides selectively towards the infected cells. Early data on the incorporation of tritiated nucleic acid precursors by Babesia-infected erythrocytes indicated that purine salvage

pathways were the only mechanism of providing purines for metabolism in the infected cells, whilst pyrimidines were synthesised de novo [6,7,14,41, 42]. Recently, the enzymes required for the interconversion of purine nucleosides to nucleotides have been measured in extracts of Babesia divergens [43]. In this paper, additional evidence has been provided that nucleoside transport sites are induced in the bovine erythrocyte by the intracellular presence of the Babesia parasite and preliminary experiments suggest that cytotoxic nucleosides, such as tubercidin, are effective as chemotherapeutic agents against B. bovis in in vitro cultures.

Acknowledgements I wish to thank Dr. I.G. Wright, C.S.I.R.O. Indooroopilly, Queensland, for his support and interest in this work and for supplies of B. boris infected blood and Prof. W.J. O'Sullivan and Dr. A.S. Bagnara (U.N.S.W.) for their advice. The excellent technical assistance of Mr. Andrew Wood with the binding studies is gratefully acknowledged. This work was supported by a grant from the National Health and Medical Research Council of Australia.

References 1 James, M.A., Montenegro,-James, S., Fajfar-Whetstone, C., Montaelegre, F., Erickson, J. and Ristic, M. (1987) Antigenic relationship between Plasmodium falciparum and Babesia boris: reactivity with antibodies to culture-derived soluble exoantigens. J. Protozool. 34,328-332. 2 Wright, I.G., Goodger, B.V. and Clark, I.A. (1988) Immunopathophysiology of Babesia bovis and Plasmodium falciparum infections. Parasitol. Today 4, 214-218. 3 Mahoney, D.F. (1967) Bovine babesiosis: preparation and assessment of complement fixing antigens. Exp. Parasitol. 20, 232-241. 4 Sherman, I.W. (1988) Mechanisms of molecular trafficking in malaria. Parasitology 96, s57-s81. 5 Irwin, A.D. and Young, E.R. (1977) Possible in vitro tests for screening drugs for activity against Babesia and other blood protozoa. Nature 269, 407-409. 6 Irwin, A.D. and Young, E.R. (1979) Further studies on the uptake of tritiated nucleic acid precursors by Babesia spp. in cattle and mice. Int. J. Parasitol. 9, 109-114. 7 Irwin, A.D., Young, E.R. and Purnell, R.E. (1978) The in vitro uptake of tritiated nucleic acid precursors by Ba-

besia spp. in cattle and mice. Int. J. Parasitol. 8, 19-24. 8 Conrad, P.A. (1986) Uptake of tritiated nucleic acid precursors by Babesia bovis in vitro. Int. J. Parasitol. 16, 263-268. 9 Sherman, I.W. (1984) Metabolism. In: Antimalarial Drugs, Handbook of Experimental Pharmacology (W. Peters and W.H.G. Richards, eds.), pp. 31-81, Springer-Verlag, Berlin-Heidelberg. 10 Gero, A.M., Bugledich, E.M.A., Paterson, A.R.P. and Jamieson, G.P. (1988) Stage-specific alteration of nucleoside membrane permeability and nitrobenzylthioinosine insensitivity in Plasmodium falciparum infected erythrocytes. Mol. Biochem. Parasitol. 27, 159-170. 11 Gero, A.M., Scott, H.V., O'Sullivan, W.J. and Christopherson, R.I. (1989) Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites. Mol. Biochem. Parasitol. 34, 87-98. 12 Young, J.D. and Jarvis, S.M. (1983) Nucleoside transport in animal cells. Review. Biosci. Rep. 3,309-322. 13 Mahoney, D.F. and Wright, I.G. (1976) Babesia argentina: immunization of cattle with a killed antigen against

276 infection with a heterologous strain. Vet. Parasitol. 2, 273-282. 14 Gero, A.M., O'Sullivan, W.J., Wright, I.G. and Mahohey, D.F. (1983) Enzymes of pyrimidine biosynthesis in Babesia bovis and Babesia bigemina. Aust. J. Exp. Biol. Med. Sci. 61,239-243. 15 Richards, W.H.G. and Williams, S.G. (19731 The removal of leucocytes from malaria-infected blood. Ann. Trop. Med. Parasitol. 67, 249-250. 16 Paterson, A.R.P., Harley, E.R. and Cass, C.E. (1984) Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method. Biochem. J. 224, 1001-1008. 17 Gati, W.P., Stoyke, A.F.-W., Gero, A.M. and Paterson, A.R.P. (1987) Nucleoside permeation in mouse erythrocytes infected with Plasmodium yoelii. Biochem. Biophys. Res. Commun. 145, 1134-1141. 18 Duggleby, R.G. (1981) A non-linear regression program for small computers. Anal. Biochem. 110, 9-18. 19 Kutner, S., Breuer, W.V., Ginsburg, H. and Cabantchik, Z.I. (1987) On the mode of action of phlorizin as an antimalarial agent in in vitro cultures of Plasmodium falciparum. Biochem. Pharmacol. 36, 123-129. 2(/ Ford, D.A., Sharp, J.A. and Rovetto, M.J. (1985) Erythrocyte adenosine transport: effects of Ca 2~ channel antagonists and ions. Am. J. Physiol. 248, H593-H598. 21 Striessnig, J., Zernig, G. and Glossmann, H. (1985) Human red-blood-cell Ca2--channel-antagonist binding sites. Eur. J. Biochem. 150, 67-77. 22 Jarvis, S.M., Hammond, J.R., Paterson, A.R.P. and Clanachan, A.S. (1982) Species differences in nucleoside transport. Biochem. J. 208, 83-88. 23 Aronow, B., Kaur, K., McCartan, K. and Ullman, B. (19871 Two high-affinity nucleoside transporters in Leishmania donovani. Mol. Biochem. Parasitol. 22, 29-37. 24 E1 Kouni, M.H., Diop, D. and Cha, S. (1983) Combination therapy of schistosomiasis by tubercidin and nitrobenzylthioinosine 5'-monophosphate. Proc. Natl. Acad. Sci. USA 80, 6667-6670. 25 El Kouni, M.H., Knopf, P.M. and Cha, S. (19851 Combination therapy of Schistosoma japonicum by tubercidin and nitrobenzylthioinosine 5'-monophosphate. Biochem. Pharmacol. 34, 3921-3923. 26 Ogbunde, P.O.J. and Ikediobi, C.O. (1982) Effect of nitrobenzylthioinosinate on the toxicity of tubercidin and ethidium against Trypanosoma gambiense. Acta. Trop. 39, 219-224. 27 Harris, D.I., Beechey, R.B., Linstead, D. and Barrett, D. (1988) Nucleoside uptake by Trichornonas vaginalis. Mol. Biochem. Parasitol. 29, 105-116. 28 Jarvis, S.M., Hammond, J.R., Paterson, A.R.P. and Clanachan, A.S. (1983) Nucleoside transport in human erythrocytes: a simple carrier with directional symmetry in fresh cells, but with directional asymmetry in cells from outdated blood. Biochem. J. 210,457-461.

29 Ginsburg, H. and Stein, W.D. (19871 Biophysical analysis of novel transport pathways induced in red blood cell membranes. J. Membr. Biol. 96, 1-10. 30 Homewood, C.A. and Neame, K.D. (1974) Malaria and the permeability of the host erythrocyte. Nature 252, 718-719. 31 Kutner, S., Ginsburg, H. and Cabantcbik, Z.I. (1983) Permselectivity changes in malaria (Plasmodium falciparum) infected human red blood cell membranes. J. Cell. Physiol. 114, 245-251. 32 Elford, B.C., Haynes, J.D., Chulay, J.D. and Wilson, R.J.M. (1985) Selective stage-specific changes in the permeability to small hydrophilic solutes of human erythrocytes infected with Plasmodium falciparum. Mol. Biochem. Parasitol. 16, 43-60. 33 Elford, J.C. (1986) L-glutamine influx in malaria-infected erythrocytes: a target for antimalarials. Parasitology Today, 2, 309-312. 34 Ellory, J.C., Elford, B.C. and Newbolt, C.I. (1988) Introductory remarks: transport mechanisms across cell membranes. Parasitology 96, 55-59. 35 Kutner, S., Breuer, W.V., Ginsburg, H., Aley, S.B. and Cabantchik, Z.I. (1985) Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development. J. Cell. Physiol. 125, 521-527. 36 Paterson, A.R.P., Jakobs, E.S., Ng, C.Y.C., Odegard, R.O. and Adjei, A.A. (1987) In: Topics and Perspectives in Adenosine Research (Gerlach, E. and Becket, B.F., eds.), pp. 8%101, Springer-Verlag, Berlin-Heidelberg. 37 Belt, J.A. and Noel, D.L. (1985) Nucleoside transport in Walker 256 rat carcinosarcoma and $49 mouse lymphoma cells: differences in sensitivity to nitrobenzylthioinosine and thiol reagents. Biochem. J. 232,681-688. 38 Aronow, B., Allen, K., Patrick, J. and Ullman, B. (1985) Altered nucleoside transporters in mammalian cells selected for resistance to the physiological effects of inhibitors of nucleoside transport. J. Biol. Chem. 260, 6226-6233. 39 Anderson, O.S., Finkelstein, A., Katz, I. and Cass, A. (1976) Effect of phloretin on the permeability of thin lipid membranes. J. Gen. Physiol. 67, 749-771. 40 Ginsburg, H., Krugliak, M., Eldelman, O. and Cabantchik, Z.I. (1983) New permeability pathways induced in membranes of Plasmodium falciparum infected erythrocytes. Mol. Biochem. Parasitol. 8, 177-190. 41 Gero, A.M. and Coombs, G.H. (1982) Pyrimidine biosynthetic enzymes in Babesia hylomysci. Int. J. Parasitol. 12, 377-382. 42 Holland, J.W., Gero, A.M. and O'Sullivan, W.J. (19831 Enzymes of de novo pyrimidine biosynthesis in Babesia rodhaini. J. Protozool. 30, 36-40. 43 Hassan, H.F., Phillips, R.S. and Coombs, G.H. (1987) Purine-metabolizing enzymes in Babesia divergens. Parasitol. Res. 73, 121-125.