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29 Buss, J.E. and Sefton, B.M. (1985)J. Virol. 53, 7-12 30 Hancock, J.F. et al. (1989) Cell 57, 1167-1177 31 Schmidt, M.F.G. and Schlesinger, M.J. (1979) Cell 17, 813-819 32 Glenn, J.S. et al. (1992) Science 256, 1331-1333 33 Van Etten, J.L., Lane, L.C. and Meints, R.H. (1991) Microbiol.

Rev. 55,586-620 34 Veit, M. etal. (1990) Virology 177, 807-811 35 Resh, M. (1990) Oncogene 5, 1437-1444 36 Moscufo, N., Simons, J. and Chow, M. (1991)J. Virol. 65,

2372-2380 37 Rhee, S.S. and Hunter, E. (1987)]. Virol. 61, 1045-1053

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8655-8659

The wily ways of a parasite: induction of actin assembly

by Listeria Lewis G. Tilney and Mary S. Tilney

T o us it is fascinating to examine the diversity of biological organisms on

the planet and how they func- tion. Many of these organisms are pathogenic and these, al- though often insidious in their behavior towards their hosts, have evolved a variety of 'special mechanisms' to enter, feed, replicate and spread that are entertaining to any biol- ogist. Listeria is no exception and to try to unravel how it operates is a fantastic trip on its own.

The intracellular pathogen Lister& has a spectacular mode of transport within and between host cells. BY inducing host cell

actin to assemble from its surface, the bacterium forms a tail composed of many short, crossbridged actin filaments. With this tail Listeria is propelled across the cytoplasm like a comet streaking across

the sky. Here we discuss the antics of Listeria and some of the bacterial genes

instrumental in maintaining it in the host.

L.G. Tilney and M.S. Tilney are in the Dept of Biology, Leidy Laboratory, University of

Pennsylvania, Philadelphia, PA 19104-6018, USA.

Listeria is a common pathogen throughout the world, and one that is thought to be carried by many of us without any obvious effects; but when Listeria reaches certain levels in the body it can be, and often is, fatal. Particularly susceptible are pregnant mothers, their foetuses, and immunocompromised individuals. Apart from its importance as a pathogen, Listeria is attractive to investigators because it provides a relatively simple system to probe cytoskeletal function in actin-based motility. The movement of Listeria is, for example, considerably less complex than the movement of an entire cell such as a neutrophil or tissue culture cell, or even a growth cone, as no membranes are involved. It is, in essence, a particle that is moving around the cytoplasm.

Shortly after Listeria makes contact with a macrophage it becomes phagocytosed (Fig. 1). Once inside the phagosome, Listeria secretes hemolysin I

and phospholipase C (Ref. 2). These enzymes break down the phagosomal membrane so that the bacterium can enter the macrophage cytoplasm, where it grows and divides using host cell nutrients. At the same time it assembles from its surface numerous short actin filaments, which form a polar tail up to 5 lam long (Fig. 1). Some 2.5 h after infection, Listeria begin to migrate around the cyto- plasm of the cell at speeds proportional to the length of

their actin tails3: up to 1 lim s -1. They always migrate head first, like a comet streaking across the sky. When the Listeria make contact with the plasma membrane of the macrophage a protruberance is generated (Fig. 1), which can be up to 40 ~tm long 4. The membrane is applied tightly around the Listeria and its tail, like a finger in a rubber glove. What hap- pens next is remarkable: when this long pseudopod makes contact with a neighboring macrophage, the second macrophage phagocytoses the pseudopod of the first. Thus within the second macrophage is a phagosome containing the plasma membrane that covered the pseudopod of the first macrophage, with- in which is the Listeria and its tail (Fig. 1). The doubly encapsulated Listeria escapes into the cyto- plasm by dissolving both membranes, again mediated by phospholipases and hemolysin, and the cycle is repeated.

© 1993 Elsevier Science Publishers Ltd (UK) 0966 842X/93/$06.00

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Fig. 1. The antics of Listeria in macrophages. Modified from Ref. 5.

Once Listeria has entered the cytoplasm of a macrophage, it can spread from cell to cell without ever again leaving the cytoplasm and entering an extracellular compartment. Thus this insidious beast cleverly avoids any detection by circulating anti- bodies. Although the reason why circulating antibodies have no effect was not appreciated until recently s, the fact that circulating antibodies are ineffective in lis- teriosis has been known for many years 6. Accordingly, this pathogen has been studied extensively as an ex- ample of a disease combated by cell-mediated mech- anisms such as CD8 ÷ lymphocytes or cytotoxic T cells (CTLs). Furthermore, since Listeria spreads from cell to cell via phagosomes, cytokines such as7 interferon can slow its spread, presumably by increasing super- oxide production in the phagosomes 7. Since cell- mediated immunity requires presentation of the major histocompatibility complex type I (MHC class I) with a fragment of the bacterial antigen by infected cells to CD8 ÷ cells, many studies have used Listeria to investigate CTL killing of infected cells 8'9.

An interesting offshoot of the role that Listeria is playing in revealing for us aspects of cell-mediated immunity is its potential as a delivery system for foreign proteins or peptides into the cytoplasm of macrophages; portions of the foreign proteins could then be processed and carried to the surface in the jaws of MHC class I. For example, when a plasmid containing the gene for 13 galactosidase attached to a listerial promoter, which induces hemolysin and other molecules (see below), was introduced into Listeria and the Listeria allowed to infect a mouse, a CD8 ÷ response was elicited against a portion of 13 galactosidase 1°. Of course Listeria is a potentially dangerous pathogen, but mutants are now available that grow and divide normally in cells but are four to five logs less virulent than wild-type organisms H. Thus mutant Listeria with the appropriate plasmid may provide a nontoxic delivery system for vacci- nating organisms to respond to future challenge by bacterial parasites that hide in the cytoplasm of cells. Listeria, then, is a spectacular model to study the

processing of a bacterial protein delivered into the cytoplasm.

Cell biological processes Viruses and/or viral proteins, such as Semliki forest and vestibular stomatis virus, have provided us with tools to dissect membrane-associated events such as endocytosis, exocytosis, secretory pathways and lyso- somal activities. Likewise, bacterial pathogens and mutants of Listeria may be exceedingly useful in the future in helping us uncover aspects of how the cyto- plasm functions. Particularly relevant to this review is that Listeria is an important model for understanding actin assembly in vivo and how the filaments thus formed may induce particle movement in cells. In fact it has been stated 3 that more is currently known about the actin antics associated with Listeria than those of pseudopods or lamellopodia of cells, such as growth cones, Dictyostelium or neutrophils, that move over a substrate. There are other cytoplasmic events that can potentially be 'dissected' using Lis- teria, including how induction of a protruberance occurs (see Fig. 1) or how a pseudopod of a cell is recognized by another cell.

Successful host colonization Listeria can grow and multiply extracellularly, for example in milk products and silage, as well as being an intracellular invader. Thus one might expect that to change environments and, more particularly, to become an intracellular pathogen, Listeria would have to turn on a number of genes to 'take advan- tage' of the intracellular environment. In essence it must adapt to the environment within the host by modifying a number of key processes so that it can grow, multiply and spread. Thus, it would have to secrete enzymes to escape from the phagolysosome, induce polymerization of actin from its surface, which it presumably does by gluing to its surface host actin filament nucleators and/or inducers of actin assembly (see below), induce the formation of a pseudopod, and put on the surface of the pseudopod some- thing that is particularly tasty to another host cell. Although we have no idea as to the number of genes necessary for a Listeria to prosper as an intracellular pathogen, and it is presumptuous to predict from the limited knowledge we have, it is theoretically possible to carry out all of these modifications with less than a dozen genes. Everything else would be supplied by the host cell and by the processes going on in the host cell cytoplasm.

There is a region of the bacterial genome contain- ing eight open reading frames, all of which seem to be regulated by a product (so far unidentified) of the so-called prfA gene 12'13. Using deletion mutants, infor- mation is available as to the identity and at least par- tial function of five of these eight genes. These include many of the 'essential modifying' components just mentioned. Thus, to escape from the phagosome Listeria secretes hemolysin 1 and phosphatidylinositol specific phospholipase C (Ref. 2); to induce actin assembly Listeria uses the actA gene product TM, and

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to escape from the phagosome of the second macro- phage a metaloprotease and phospholipase C are used is. Since four of these five proteins are enzymes, their characteristics - substrate specificity, optimal conditions for activity, cofactors and so on - are being explored. Furthermore, how these five proteins are regulated is partially understood 16.

Formation of the actln tail Within 15 min after the Listeria have escaped the phagosome and entered the cytoplasm, they begin to be surrounded by a thin cloud of actin filaments. One hour later many have short tails, by 2 h many more have short tails and some have longer tails, and by 3 h after the initial infection some have very long actin tails and are moving around in the cytoplasm as a prel- ude to entering a pseudopod and spreading s. From these static observations it might be predicted that the length of the tail is proportional to the time the Lis- teria has spent in the cytoplasm 17. Because filaments do not assemble on newly formed surfaces such as those resulting from septation (Fig. 2) and since septa form near the center of the elongated bacterium, each ensuing division amplifies the polar distribution of actin filaments so that after four divisions some Lis- teria have a tail up to 5 gm long (Fig. 1). Thus we began to suspect that the older the surface of the Listeria, the longer its attached tail will be. This prediction could be tested by examining the length of the tails of all the Listeria that were present in the cytoplasm of a macrophage after four divisions. If, indeed, the age of the bacterial surface is related to tail length, then one would predict (see Fig. 3) that after four divisions there would be one with a long tail, one with an in- termediate length tail, two with short tails, and four with no tails, only clouds. This has been experimen- tally verified 17. These observations, made on fixed cells in which we examined the net growth of the tail of Listeria as it resides in the cytoplasm, are not in- consistent with video observations made by ourselves and others TM. These show that, upon occasion, indi- vidual tails can rapidly break down and then re-form, and that there is a continual flux of actin subunits into and out of the tail (see below).

The next question we should address is how the actin filaments rearrange to form a smooth contoured tail extending from only one end of the Listeria. The key, of course, comes from observations on the div- ision of Listeria into two daughters, in which each daughter cell has a newly formed end, bare of actin filaments (Fig. 2). To form a tail, all that is necessary is for the existing actin filaments to become inter- connected and as progressively more filaments are formed, a tail will naturally develop simply by the mechanics of growth. Two lines of evidence indicate that the actin filaments in the tail are crosslinked. The first is the observation that Listeria with attached tails (up to 5 I.tm in length) can be isolated following detergent extraction19; the component filaments do not drift away, nor do they become rearranged even if the tails are left for more than 1 h at room tempera- ture (Fig. 4). This is particularly significant as the

Flg. 2. Thin sections through Listeria in the cytoplasm. (a) A Listeria with a short tail. Actin filaments extend from the lateral and basal surfaces of the bacterium but are conspicuously absent from the anterior pole (arrow). (b) A Listeria in the final process of division. Whereas actin filaments extend from all surfaces of this dividing cell, they are sparse in the area where septation occurs. Notice that the right half of the Listeria has a thicker actin cloud and a more extreme tail than the left half. (¢) A newly divided Listed& Where division has just occurred the surfaces of the Listeria are free of actin filaments. The Listeria on the right has a much longer tail than the one on the left. Reproduced from Ref. 17, with permission.

maximum length of the individual filaments making up the tail does not exceed 0.3 lam. Second, using immunofluorescent techniques, the tails are seen to contain the crosslinking protein ot actinin 4. Further details on the shape and consistency of the tail are presented by Tilney et al.19, including the fact that the tail appears 'hollow' in section with a higher density of filaments at the periphery and few in the center (this again just reflects the mechanics of growth).

Polymerization of actin from the bacterial surface Before describing details of the polymerization of actin induced by Listeria, we should mention three facts about the assembly of actin in vitro. First, actin filament assembly occurs in two stages: in stage I it is thought that three monomers come together 2° to form a trimer, in a process called nucleation; in stage 2, monomers add to the ends of the trimer or nucleus and the filament elongates. Nucleation in vitro is a

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©

. 0 ,.Z'.-'L

( ~ ~ ~ : ( .... ~ ,----

( ~, 4 ; CQ (z3

Fig. 3. The lineage of a Listeria that had entered the cytoplasm of a macrophage. Since the Listeria divides every 50 min, by 4 h it should have divided three times (which includes 45 min for the Listeria to escape from the phagosome and enter the cytoplasm). After four divisions there should be 16 Listeria, two with a long tail, two with a shorter tail, four with a tiny tail, and eight with no tail. Reproduced from Ref. 17, with permission.

newly assembled filaments were uni- 0:00 directionally polarized with their

barbed ends associated with the surface of the bacterium (Fig. 5). Furthermore, since the critical concentrations for the

0:50 polymerization of actin are different for the two ends, we used actin concen- trations below the critical level for the

J:40 pointed end to show that the filament elongation must be occurring by monomers adding to the barbed ends, which are the ends associated with the

z:30 surface of Listeria. Putting all this t o g e t h e r we 23 concluded that Listeria induces the assembly of actin from its

3:z0 surface by secreting a nucleator. This conclusion is probably incorrect

for two reasons. First, mutants of Lis- teria that lack the actA gene are unable to assemble actin filaments on their sur- faces in vivo TM, but, curiously, mutants that express increased amounts of actA on their surfaces do not nucleate increased numbers of actin filaments from their surfaces in vitro. Second, and

more telling, are the observations of Heuser and Morisaki 24. They showed that by treating macro- phages with a combination of lanthanum and zinc, endosomes can be induced to move around the cell with an actin tail similar to that attached to Listeria. Thus the formation of an actin tail does not require a contribution from Listeria, but is a characteristic of the host cell that can be induced by bizarre conditions or infection by Listeria.

What then does actA do, and how does Listeria induce actin filament assembly from its surface? The easiest explanation, and one that is consistent with the remarkable behavior of endosomes, is that actA is a 'glue' that attracts to it host cell molecules that stimulate and/or nucleate actin assembly. The surface of endosomes, when the host cell is treated with lanthanum and zinc, would seem to attract the same molecules in a similar way. The nature of these host molecules is under intense scrutiny now in several labs. One possibility is that, because of proline-rich repeats in its primary structure TM, actA is binding an actin-binding protein. Similar polyproline repeats are found in vinculin, another actin-associated protein TM. Using this information, Theriot and Mitchison 2s sug- gested that actA may bind profilin, and by immuno- fluorescence they showed that this is indeed the case. From these observations they suggested that profilin may facilitate actin assembly at the surface of Listeria by increasing nucleotide exchange and thus the local concentration of polymerizable actin or actin bound to ATP. Unfortunately this hypothesis cannot ac- c o u n t for the observations mentioned earlier which demonstrate that actin assembly in vitro takes place on the surface of Listeria, because the actin used in those experiments in vitro was ATP actin 23.

When we first examined the actin filaments in the tail of Listeria, we were struck by the fact that the ilia-

Fig. 4. Thin section through a macrophage that has been extracted with the detergent Triton X-IO0. The filaments making up the tail of the Listeria are stable for many hours after detergent ex- traction and they remain connected (crossbridged) to- gether. Bar represents 1 ~tm. Reproduced from Ref. 17, with permission.

chancy event and thus rate limiting; in contrast, once nucleated, elongation occurs rapidly. Thus, in cells there are likely to be proteins that nucleate actin as-

sembly. Second, actin filaments are polar, which can be easily seen in electron micrographs by examining filaments that have been decorated with subfragment 1 of myosin. What one sees is an 'arrowhead' configuration with 'barbed' and 'pointed' ends to the filaments. Third, the mini- mum concentration of monomers (critical concentration) that will permit elongation is different for the two ends, being 0.1 IIM for the barbed or high-affinity end, and 0.7-0.9 }IM for the pointed end 2°-22.

We have demonstrated that something attached to the surface of Listeria induces actin filament assembly 23. In our experiment, macrophages were infected with Listeria and then cytochalasin was added to inhibit actin as- sembly. Three hours later the macrophages were extracted with detergent and exogenous G actin was added to the preparation under polymerizing conditions. What happened was that each Listeria resembled a tiny star with actin filaments extending from its surface 23. Subsequent analysis using subfragment 1 of

• 19 myosin demonstrated that the

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ments that make up the tail are all short, seldom ex- ceeding 0.2 ~tm. This observation was unexpected as one would imagine that the tail would be made up of a parallel bundle of actin filaments like those in the acrosomal process of Thyone sperm 26 or those in a microvilhs 27'2s. By decorating the filaments in the tail with subfragment 1 of myosin we 19 showed that, even though short, the pointed ends of the filaments in- variably are oriented towards the tip of the tail (Fig. 5). Putting these two observations together we pre- sume that the tail grows by the nucleation of actin from the surface of the bacterium and the subsequent crossbridging of the elongating filament to neighbor- ing filaments. The elongating filament is released from the surface of the bacterium and a new one is nucleated and crossbridged and so forth. The ques- tion then becomes, what is controlling the short length of the filaments and why is the tail built in this way?

At this point all we can do is to speculate, as we have only limited information on exactly what the host cell cytoplasm is doing and why it is doing it. What we think is occurring is that the host cell has a substantial store of unpolymerized actin, which has been estimated to be 100-200 ktm (Refs 26, 29). To control spontaneous assembly the host must be sup- pressing extraneous actin filament nucleation and growth using a variety of actin-binding proteins. We propose that, to overcome the host controls, the bac- terium secretes on its surface the protein actA which, by binding to other host proteins, generates sites of nucleation that initiate actin assembly. The host cell stops the elongation of the filaments by using 'barbed end cappers' such as cap Z (Ref. 30) or gelsolin3L At this point the bacterium would be out of business but it avoids this problem by releasing the short recently assembled filaments and nucleating new ones that are, in turn, terminated by the host. The result is a tail of many short filaments that gradually increases in length by the nucleation of more filaments. A similar proposal has been recently suggested by Theriot and Mitchison 32 for fibroblast motility.

How Listerla move A number of investigators have become fascinated by the movement of Listeria in the cytoplasm of eucary- otic cells 3'4,14,18'33,34 and it has been studied by a num- ber of techniques. Given the polarity of the actin fila- ments in the tail and the fact that Listeria always moves like a comet with the bacterium leading and the tail trailing behind, conventional myosin (myosin II) located in the cytoplasm proper could not account for the movement because the polarity of the actin filaments is inappropriate. It would be as if skeletal muscle actually elongated during contraction. Hav- ing eliminated this possibility, we can think of three possible explanations for the movement. The first re- quires that the assembly of actin itself (and the release of these newly assembled filaments) provides a propulsive force comparable to that suggested for the elongation of the acrosomal process of Thyone sperm 27. The second invokes a motor protein, such as

Fig. 5. On the left is a thin section through a Listeria with a tail whose actin filaments have been decorated with subfragment 1 of myosin. The fi laments are all short, not more than 0.3 lam long, and the lengths of the filaments at the tip and base are comparable. On the right is a light print on which we have indicated in ink those filaments whose polarity we can unequivocally determine from the left-hand micrograph. Note that in all cases the filaments have their pointed ends nearest the tip of the tail and their barbed ends nearest the Listeria. Reproduced from Ref. 17, with permission.

a myosin which is attached to the surface of the bac- terium; the action of this motor must be coupled to the assembly of actin. The third is the use of a cyto- plasmic motor that operates in the reverse direction to myosin. Since no such reverse motor has thus far been described, we do not know how seriously to take this possibility. We will therefore confine our discussion to the first two possibilities.

The most dramatic evidence for the first possibility comes from an experiment in which G actin covalently coupled to caged resorufin (CR) was injected into a Listeria-infected potoroo kidney epithelial (PtK2) cell line 3. CR actin is nonfluorescent and is incorporated readily into actin-containing filaments. Upon illumi- nation with ultraviolet light the CR is readily and efficiently converted to the bright red fluorescent parent compound, resorufin. Using this probe, short

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Rg. 6. The spread of Listeria from one macrophage to'another. Extending from the macrophage at the base of the micrograph is a pseudopod containing a Listeria near its tip. A second macrophage, above, is phagocytosing the pseudopod of the first macrophage. Bar represents I pm.

segments of the Listeria tail could be photoactivated and, by combining the fluorescent image with that of the moving bacterium by phase contrast video mi- croscopy, it was shown that the photoactivated mark on the tail remained stationary in the cytoplasm as the bacterium moved away from the mark. It was thus concluded that the filaments must appear (assemble) continuously at the bacterial surface and be released from the bacterium as it moves on, so the rate of bacterial movement is equal to the rate of actin filament appearance. Furthermore, the length of the tail is linearly proportional to the rate of movement. Putting these two observations together, it was con- cluded that the motility of Listeria involves the con- tinuous polymerization and release of actin filaments at the bacterial surface, and that the rate of filament generation is related to the rate of movement 3. Thus the data are consistent with actin polymerization pro- viding the driving force for propulsion. A similar con- clusion was reached by Sanger e t al. 18'34, who halted listerial propulsion by cytochalasin D treatment, and re-initiated it by microinjection of rhodamine- labelled monomeric actin.

The second possibility, in which myosin molecules attached to the surface of the bacterium induce move-

ment, is also conceivable although there is no evi- dence to support it. In this model the bacterium would be continuously moved forward relative to its associated short filaments. What is essential in this model is coupling of myosin motor antics with the assembly and disassembly of actin filaments.

Function of the actln cytoskeleton of Listerla One might assume that the major function of the actin tail is to move the Listeria to the cell surface as a prelude to dispersal. This is certainly reasonable. However, equally important is the use of the actin tail in pseudopod formation. This is essential for spread- ing because the Listeria must end up in the tip of a pseudopod in order to be phagocytosed by a neigh- boring host cell (Fig. 6). Since projections of animal cells such as microvilli, microspikes, pseudopods and stereocilia all contain a core of actin filaments necess- ary for growth and maintenance of these asymmetric processes, it seems reasonable to conclude that the actin tail of Listeria could provide a core for the gen- eration of a pseudopod. All that is required is for the actin filaments at the margins of the tail to bind to the plasma membrane and by zippering these actin fila- ments to the plasma membrane, a pseudopod would be generated with the Listeria at the tip. In microvilli, the connections that extend from the lateral surface of the filament bundle to the membrane are myosin I (Refs 35-37). It is undoubtedly not an accident that the polarity of the actin filaments in all these ceil'ex- tensions, such as microvilli, microspikes, stereocilia and listerial tails, is identical (barbed ends located at the tips of the process) 2s'29. The common feature is that this polarity may regulate the attachment of~ these connections to the plasma membrane; this in turn allows formation of a projection which, in the case of Listeria, has the bacterium at its tip.

More questions In our review we have left many unanswered ques- tions. Some revolve around the control and function of the eight genes turned on by the single promoter that is induced by prfA. Others have to do with ident- ifying the components 'donated' by the host that allow Listeria to form actin tails, to move, and to enter and bind to the inner surface of the membrane of a pseudopod. And still others are concerned with how Listeria regulates the expression of the genes essential for surviving in the cytoplasm.

Although these are interesting problems, to us there are two questions that appear to be the most important in developing an understanding of the biology of Listeria. The first is the general question of what is the minimum number of genes that must be expressed by Listeria in order for it to become an in- tracytoplasmic parasite. Are they only the eight genes turned on by prfA, or are there others, some of which may be regulated by other inducers? The second is, how does a macrophage located adjacent to a Lis- teria-infected neighbor recognize and phagocytose a pseudopod containing a Listeria? What makes it tasty? Is it a product synthesized and somehow put on

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the cell surface by the Lis ter ia or is it a host molecule that the Lis ter ia has somehow induced the host cell to concentrate on the tip of the pseudopod?

Acknowledgement Supported by a grant from the National Institutes of Health HD 14474.

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In file next issu e o f in M ology

In the o d ~ journals A selection ~of recently ~ t i s h e d articles of interest to T/M reack~rs

• phenotl~pt¢ switching in Canaidaalbicans, by D.R. Soil, B. Morrow and T. Srikantha - Trends/n Gene~Jcs 9 (2), 61 -65

=The role of host Ohospt'mrylation In~bacteri~ O ~ ~ i s , by J.B. ~ s k a ~ S. F a I ~ - Trends in 9 (3), 8 5 - 8 9

. . . . . . . . ~ ~ k ~ , 3 (2), ~ 5

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• Secretory transport in P/asmod/um, by H.G. Elmendorf .and K. Haldar - Paras TOa~ 9 (3), 98 -102

The May issue of Parasltolo~ Todaywill ~ U S ~ drug r e s i ~ in parasitic s.

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