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Helminth neuromuscular systems
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Helminth Neuromuscular Systems A.G. Mat,le and T,G. Geary London, UK September 1996 The r,rog,-ar~rne for this specia~ symposium entitled 'The molecular biochemistry and physiology of helmin.th neuromuscular systems' encompassed a variety of research topics on helminth neuromuscula/ function, involving researchers from a variety of disciplines. The two-day symposium was divided into five sessions of plenary lectures, two sessions for shorter talks, and a poster session (a total of" 40 presentations), l'he aim of the meetin~ was to help synthesize current uT,derstanding and recent developments in the study of the nervous and muscular systems of helmlnths. One underlying theme for the meeting was the concept that neuromuscular systems of helminths contain a plethora of known and putative drug targets. A more complete understanding of the basic principles and molecular biology behind integrated and coordinated helminth motor activity is likely to prove crucial to future directed strategies for anthelmintic discovery and parasite control. The s/mposium, which attracted some 100 national and international delegates, was started by John Stephenson (the Wellcome Trust, London, UI0, who outlined the long-term commitment of the Wellcome Trust to parasitology research. The figures presented indicated that parasitologists did rather be~er in gleaning funding from the Wellcome Trust (on a percentage basis) than did mo~ other areas of research. While the audience was appropriately grateful, a common sentim~nt was, 'heaven help the other areas!'.
components, their ~rimitive nervous systems offer unique opportun~iPs to gain insights into neuromuscular functl~:~ It is clear that, although comparatively simple, helminth neuromuscular systems are very complex and are likely to oossess most of the major types of signaling pathway that have been identified in higher organisms.
Platyhelm~nth Nervous Systems A detailed discussion of the structure and chemical complexity of the nervous system of pla~,~e!minths as revealed by immunocytochemistry interfaced w~th confocal imaging technolo~ was encompassed in a presentation by D.W. Halton (The Queen's University of Belfast, UK). The nervous system of platyhelminths plays a pivotal role, not only in the coordination of motor behavior, but also, through neurosecretory processes, in growth, differentiation and development. Although the information provided by immunocytochemical studies is usually restricted to antigen Iocallzztlon, changes in peptide immunostalning p~e,~,~ and intensities in the reproductive system of selected trematodes were shown to be associated with reproductive activity. These studies demonstrated that ICC could be successfully applied to demonstrate specific functions for neuropeptides (in this case, FPIRFamide-related peptides or FaRPs) in a physiological process. The molecular physiology and pharmacology of plat,/helminths was discussed by M.C. Jeziorsky (The Whitney Laboratory, Florida State UniversW, USA) and R.A. Pax (Michigan State University, USA). Our understanding of
Evolution of Helminth Classical Transmitter and Neuropeptide Si~n~lin~ Systems
neurorr,uscuiar function in f1~t'~,emqs is currently limited, and mos-t of what is known is confined to the trematode, Schistosoma mansoni and the turbellarian, Bdelloura condido. Several flatworm
Two elegant presentations provided an overview of the evolution of classical transmitter molecules and neuropeptides and set the scene for the syrr,posium (EJ. Walker Southampton University, Ul~ and C. Shaw, The Queen's University of Belfast, UIC). Since the platyhelminths are the basal stock of the deuterostomian line, and one of the eadiest groups to have developed functional central and peripheral neural
neuromuscu!ar proteins have been iden•,'Jfied as potential dnJg targets, including acetylcholine (ACh), serotonin (5-H'I-) and neuropeptide receptors, as well as K+, Ca2+ and Na + channels. The emergence of channel information fi'om B. candldo should facilitate the identificatJon of analogous proteins in parasitic forms; homologous calcium channel cDNAs have been identified in both Bdelloura and Schistosoma. Other signaling path~says that were ,mpkated in
Parasitology' Today. vol. 13, no 5. 1997
Copyright © i't07 E l ~ S o ~ ¢ e L t d
platyhelminths included that of ~,e free radical gas, nit,qc oxide (A. is,dholrn and colleagues, Abo Akademic University, Finiar~d) With respect to neuropep:ides, little is known about their roe in platyhelminths and the only parasitic fo,,-m from which a neuropeptide has been structurally characterized remains the cestode, A4onlezio expanso. Neve~heless, numerous neuropeptide structures are emerging from the turbellarians and these have been shown to be active on turbellarians i (R.N. Johnston, The Queen's University. of Belfast, UK) arid on trematodes :z.3 (-F.A. Day. Michigan State Univem~, USA; NJ. Marks, The Queen's University of Belfast, U,V).,.Indeed, flatworm peptides that possess the C-terminal motif, YIRFamide, have been shown to be extremely potent in inducing contractlon of isolated muscle fibers of S. mansoni (T~. Day, pers. commun.) and remain the only known contractors of flatworm muscle fibers. Further characterization of these muscle-membrane prot,Jins is required if their po'Lential as drug targets is to be realized. Increased eff'ol'ts in a variety of scientific fields are needed to illuminate the physiological mer~,~ni~m~ that underlie platyhelminth neuromuscular funct!on. Direc'~ physiological characterization of the functior=s of endogenous neuromedlators i~l intact helminths and in =lispers.ed cell types as well as tJqe rr,olecular characterization and expression of the native channel and receptci proteins, will broaden our unc~erstamdin~ of these control systems. "]'he advancement of these goals ¢,'ill only be realized through the t,vpes of multidisciplinary approach that have been adopted at Michigan S~ate and Flodda State universkies.
Nematode Nervous Systems Current understanding of neuromuscular function in nematodes is more advanced than that of platyhelminths.A.O.W. Stretton (University, of Wisconsin, USA) outlined some of the data ge~erated in his laboratory through the many years of detailed experimentation required to devise a model of coordinated movement in the pig gastrointest:nal nematode. Ascar/,'ssuum. Furthermore, even with a basicunderstanding of the ar::hitecture
AIl~ghts re.served 0169-47SB/97/$17.C0 S01~.947S8(97;01034-X
|63
News of the nematode nervous ~ystem and of the fiJnctions of their classical neurotransmitters, the influence th.at endogenous neuropeptides have on neuromuscular function will greatly complicate ma~ters The number of identified nematode neuropeptides is increasing (alarmingly!) through the efforts of the Caenorhabditis elegans genome sequencing teams and the Chris Li group (Boston Universe,, USA). T.~e Stretton laboratol-/has embarked c~ a research programme to asce,~in ~ ~u,lc[ions of identified A. suum neuropeptides at the cellular level, His group has identified at least 20 peptides that might be called FaRPs (since identifying the first one nearly ten years ago), and has begun the physiological characterization of all of th~n. This work ilas been complemented by experimenLsaimed at understanding the transductlon pathways involved in FaRPactions on nematodes, carried out in the USA at Phan~acia and Upjohn4 (T.G, Geary, A.G, Maule, j.W. Bowman and O.P. Thompson) and in the UK at Southampton Unlversity s (R.J. Walker and I_ Holden-Dye). Nitnc oxide (NO), c~MP, Ca2+, K* and Clchannels have been implicated as signaling mechanisms for FaRPe~ic systems in A suum. Nematode FaRPs are aJso implicated in pharyngeal function in /~ suum fD.JA. Brownlee, Southampton University, UK) and were found to be active on trematode motility (N.J. Plarks, The Queen's University of Belfast, Uk3, indicating at least some inter-phyla conservation of FaRP-receptor structures. Peptidergic systems of nematodes pro-. vide anthelmintic targets which invite screen development and the implementation of drug-design prog~mmes. However, our knowledge of nematode neuropeptidergic systems is still extremely limited and should be expanded through concerted research efforts on numerous fronts. "Fne potential of the neuropeptidergic system for dru~ discovery, is not restri~ed to the signaling mechanisms (neuropeptides and receptors), but indudes re!.=~ed regu!atory systems, For example, neuropep'dde degradation and neurotransmltter uptake/transporter systems offer further opportunities for exploitation (P~E. Isaac, University of Leeds, UK). Althou&h current understanding of these systems in parasitic nematodes is limited, there are now numerous examples of cloned nematc,de transporters and neuropeptid~es from C. elegans, Rapid progress in this field could be made by transgenic manipulation of C. ei~oo-,r;s aimed ~t generatJng neu~transmitter-, receptor164
and tram,porter-gene deletion mutants and characterizing their phenotypes.
Ionotropic Channels The membrane-level electrophysiological effects of known nematlcides were described by R.J.Mar:in (University of Edinburgh, UK), working on A. suum. An exciting development has been the recent adaptation of the Kostyuk method to the ,'cco~ing of voltagedependent cunents in isolated muscle bag-cell prepal~tions. This technique enables more precise measuremehLs of voltage-activated currents and the intrace!lular application of test compounds during voltage-clamp procedures, The Kostyuk method has been used to identify a voltage-activated, ACh-sensitive, inward non-sc;~ctive cation current on the bag region of A. suum rnus,:le cells, This current is reduced by the FaRP, PFI. Application of this technique will likely advance our understanding of membrane level events in nematode neuromusculature and provide evidence for mechanisms of drug action. H. Wilkinson (M~rck Research Laboratories, New Jersey. USA) summarized the current understanding of the mode of action of the most successful class of antheimindc to date, the avermectins. Expression cloning studies on C elegans carded out at the Merck Research Laboratories have identified glutamate-activated CI- channels as important targets for the avermectin Jass of drugs, although their endogenous functions remain unknown 6. The importance of these channels is highlighted by the fact that similar channels have not been identified in mammals. Data obtained with C elegans knocko~¢ mutants indicate that a family of these channels occurs in nematodes, which may have multiple functions. Binding of ivermectin to C. elegans is reduced but not eliminated in these knock-out mutants (which have no detectab!e phenotype), su~esting that additional avermectin receptors are present~ N.S. Delaney and colleagues (Bath University, UI0 reported that receptor subunits, which are homolo~,.ous to some of the C elegans subunits, have been cloned from Heemonchus contortus and T.M. Skinl!er (also at Bath) raised antisera to a,~ inhib¢ory amino acid receptor subunit and localized immunos*,~ining to the lateral nerve cords of H. contortus and muscle bags of A. suum. On a cautionan/ note, I ~ Murray and R.J. Martin (University of Edinburgh, UK) reporleu "
that there may be differences between the avermectin receptors OfA. suum and C. elegans pharyngeal muscles, based on the fact that cloned C elegans glutamategaled Ct- channels are insensitive to L.-aspa~a;e, whereas it increases the input con&,c'tance of A. suum pharyngeal muscles. Other nematode-muscle channel proteins have been implicated as sites of action for nematicidal drugs, including levamisole. This drug acts on nicotinic acety'~choline receptors ,'nAChRs), which occur as members of a multigene family in nematodes (D.B. S~elle, University of Cambrid£e, USA) Molecular cloning methods (card_,d out at Cambridge University, Mass'~chusetts General Hospital Cancer Center and the University, of Texas at S~JnAntenio) have identified a !arge Family of C. elegens AChR subunits w:~ich are closely related to analogous i~.sect proteins. !mpor~.~ntly, ,-elated subunits have been cloned "From th~ parasitic forms, TrichostronEylus colubrifon'nis and Onchocerca vohulus. Although levamisolesensitive currents can be detected in Xenopus oocytes after the injectie~ of cRNAs encoding C. elegansnAChR subunits, expression is not robust and it is not known if these are identical to the wild-type receptor. In comparison with mammalian nAChRs, the nematode analogues display some unique pharmacological characteristics, such as their insensitivity to lophotoxin, which can be understood on the molecular level. Such chara~cristics suggest that future exploitation of parasitic nematode ionotropic receptors may be profitable for anthelmintic discovery.
Anthelmintic Resistance A most worrisome development for parasite control is the ever-increasing catalogue of drug-resistant strains. Resistance has been reported for several of the drugs which specifical',ytarget (or are believed to target) parasitic helminth neuromuscular systems including levamisole, ivermectin and praziquantel. Current knowledge of the pharmacology of anthelmintic resistance in heh,',ii,th pa~s~es was summarized by N. Sangster (University of Sydney, Australia). it is clear that detailed understanding of the mechanisms of anthelmintic resistance will shed light on how these drugs work and on the biology of the systems they target. Molecular genetic studies on C eleganshave greatly advanced our understanding of benzimidazole-resistance and are likely to be critical in further characterization of Paras:'.oloEyTc~y. vs!. !3. he. 5. !oq7
N ews resistance to neuro- and myo-actlve compounds. In th;s way, P. Hunt (University of Edinbupgh. UK) and col~eagues screened a large number of iverrnectinresistant C, elegans to identify genes which confer and suppress iverrne~in ~sistance. Un!!k~ the case in 9ara~if,_ nemato:':es, ivermectin resistance in C e/ego ~ is typically recessive. The new gen~s are at least semi-dominant, wn;ch make them of more relevance for he situation in parasites. They also pn=sented evidence that suggested that iverme~in resistance is linked to am!:}h~d-deHved pharyngeal modulation. The prospects for rational api~roaches to anthelmintlc discovery were discussed b T T,G. Gear-/(Pharm ,c;a and Upjohn Co,, USA). The concept of rational drug discover,/ was introducec ~ in the cor.text that other forms of drug discover/must, by analogy, be 'irrational', wh~cq is certainly an irrational conclusion. P.andom s~reening oroglamrnes hace bee;; funclamental in the discover,/ of all current front-line anthelmintics. Ne~er developments in the pharmaceutical industry have advanced high-throughput random screening in mechan;sm-based assays (as opposed to who!e organism toxici b' assays) to be often favoured over computationally based drug design strategies. Anthelmintic discovery programmes can adopt this same strateE/ if we gain greater understanding of the biology of these organisms. Once good
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drug targets are identified, they car, be inct ~oorated into mechanism-based screens. Achieving this understanding is absolutely dependent upon increased basic research on nematode hieing', indudir, f, neurobiology. Again, the irr,pr~tanr~ ~" the C. ele~ans £ont~m~ sequencing project to the identification of new anthelmintic ta~gets A,as emphasized.
e~.cellcn~ opportunity to form a coherent community of iike-minded sc!entists. Based on its success, plans a_-ebein~ made for a similar gathering in 1998, On behalf of the delegate'~ we wouid like to thank the organizers, ,~o¢. De,,;4 'vV Halton (The Queen'.; Unive,sity of 3elfast) and Dr P.!c%=~'. Me-tin (ErJinburgh ;Jniversity) for a highly successfvl meeting. The event was very generously sponsored by Bayer, BSP, Janssen Anima! Health, Pfizer Central Research, Pharmacia and Upjohn, The Wellcome Trust and Zeneca. All 13 of O n a Lighter N o t e the plcnar,/lectures are due to be published The conference was concluded by a in a special supplement of Parasitology '~. highly convivial Chinese banquet which was thoroughly enjoyed by all invited References I }ohr,ston.R.N. e[ al. (1996)J. Neurochem.67, sF.eakers and sessio~ chairs (except R.]. 814-821 2 Day, TA. et el. (1994) Parosito!e~y i09. 'ffartin to whom the bill was pre455-459 ~ented!). The best pos[er presentation 3 Marks. N.J. et at. (,1996) Parasitology 113. won a bottle of whisky, which went to 393-40~ Gunnar Mair (The Queen's University 4 l'4aule.,~-G.et eL (1996.1PorositoL Toda,~ 12. of Belfast. UI0 for his study of #laW35t.-357 5 Brownlee.D.J~ et aL (:996) Pcr,Tsitol. Todct;, helminth musculature using fluore.-'t~_r,'~ 12. 343-35I tags and confocal microscopy. 6 I:tter. A. et ell (t'~q~ J. 6ioL Chem. 271, 16035-16O39 Acknowledgements 7 Halton. DW. ('99) Paras;toto~/ 102, On 18-19 September 1996. a special symSLS118 8 Ha,~nn, D.W. (1996)Parositology I 13,51-5238 posium entitled 'The molecular biochemistry and ph~iolo~,;, ~f helminth neuromuscula,- systems' was held at C~.," Univer~,itv. A G ~ a I~,aule is at The Comparative NeuroLondon. This was the second , .ch s'~mF.o- endocrinology Pesearch Group, Schools of 8ioiogy and Biooh6mist~ and Clinical Medislum and followed the very successful ~bsh Society for Par~s~toloK)' (B~.;P) meet:,'b on cine, ,~heQueen's University of Belfast, ~elfast, Pa~-asite Neurobiology which was heL5 at UK BT7 JNN. Tim Gear), is at/,n,mal Health the same venue7. The sym/.,osium was w2:- Discover, Research, Pharmacia and Upjohn refuted by the recent surge ,n the numb,:r Co., Kaiamazoo, MI 49001, USA. Tel: . t ~ of laboratories working on h~Iminth ne,Jro- 12J2 272059, lax'. +44 1252 256S05, biolo.~ayon ~o,pl~i~:icatedlevels, ar# was an e-n',~/: a.nwu'~,~,_,~.a-_ uk
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Helminth Neuromuscular Systems A.G. Mat,le and T,G. Geary London, UK September 1996 The r,rog,-ar~rne for this specia~ symposium entitled 'The molecular biochemistry and physiology of helmin.th neuromuscular systems' encompassed a variety of research topics on helminth neuromuscula/ function, involving researchers from a variety of disciplines. The two-day symposium was divided into five sessions of plenary lectures, two sessions for shorter talks, and a poster session (a total of" 40 presentations), l'he aim of the meetin~ was to help synthesize current uT,derstanding and recent developments in the study of the nervous and muscular systems of helmlnths. One underlying theme for the meeting was the concept that neuromuscular systems of helminths contain a plethora of known and putative drug targets. A more complete understanding of the basic principles and molecular biology behind integrated and coordinated helminth motor activity is likely to prove crucial to future directed strategies for anthelmintic discovery and parasite control. The s/mposium, which attracted some 100 national and international delegates, was started by John Stephenson (the Wellcome Trust, London, UI0, who outlined the long-term commitment of the Wellcome Trust to parasitology research. The figures presented indicated that parasitologists did rather be~er in gleaning funding from the Wellcome Trust (on a percentage basis) than did mo~ other areas of research. While the audience was appropriately grateful, a common sentim~nt was, 'heaven help the other areas!'.
components, their ~rimitive nervous systems offer unique opportun~iPs to gain insights into neuromuscular functl~:~ It is clear that, although comparatively simple, helminth neuromuscular systems are very complex and are likely to oossess most of the major types of signaling pathway that have been identified in higher organisms.
Platyhelm~nth Nervous Systems A detailed discussion of the structure and chemical complexity of the nervous system of pla~,~e!minths as revealed by immunocytochemistry interfaced w~th confocal imaging technolo~ was encompassed in a presentation by D.W. Halton (The Queen's University of Belfast, UK). The nervous system of platyhelminths plays a pivotal role, not only in the coordination of motor behavior, but also, through neurosecretory processes, in growth, differentiation and development. Although the information provided by immunocytochemical studies is usually restricted to antigen Iocallzztlon, changes in peptide immunostalning p~e,~,~ and intensities in the reproductive system of selected trematodes were shown to be associated with reproductive activity. These studies demonstrated that ICC could be successfully applied to demonstrate specific functions for neuropeptides (in this case, FPIRFamide-related peptides or FaRPs) in a physiological process. The molecular physiology and pharmacology of plat,/helminths was discussed by M.C. Jeziorsky (The Whitney Laboratory, Florida State UniversW, USA) and R.A. Pax (Michigan State University, USA). Our understanding of
Evolution of Helminth Classical Transmitter and Neuropeptide Si~n~lin~ Systems
neurorr,uscuiar function in f1~t'~,emqs is currently limited, and mos-t of what is known is confined to the trematode, Schistosoma mansoni and the turbellarian, Bdelloura condido. Several flatworm
Two elegant presentations provided an overview of the evolution of classical transmitter molecules and neuropeptides and set the scene for the syrr,posium (EJ. Walker Southampton University, Ul~ and C. Shaw, The Queen's University of Belfast, UIC). Since the platyhelminths are the basal stock of the deuterostomian line, and one of the eadiest groups to have developed functional central and peripheral neural
neuromuscu!ar proteins have been iden•,'Jfied as potential dnJg targets, including acetylcholine (ACh), serotonin (5-H'I-) and neuropeptide receptors, as well as K+, Ca2+ and Na + channels. The emergence of channel information fi'om B. candldo should facilitate the identificatJon of analogous proteins in parasitic forms; homologous calcium channel cDNAs have been identified in both Bdelloura and Schistosoma. Other signaling path~says that were ,mpkated in
Parasitology' Today. vol. 13, no 5. 1997
Copyright © i't07 E l ~ S o ~ ¢ e L t d
platyhelminths included that of ~,e free radical gas, nit,qc oxide (A. is,dholrn and colleagues, Abo Akademic University, Finiar~d) With respect to neuropep:ides, little is known about their roe in platyhelminths and the only parasitic fo,,-m from which a neuropeptide has been structurally characterized remains the cestode, A4onlezio expanso. Neve~heless, numerous neuropeptide structures are emerging from the turbellarians and these have been shown to be active on turbellarians i (R.N. Johnston, The Queen's University. of Belfast, UK) arid on trematodes :z.3 (-F.A. Day. Michigan State Univem~, USA; NJ. Marks, The Queen's University of Belfast, U,V).,.Indeed, flatworm peptides that possess the C-terminal motif, YIRFamide, have been shown to be extremely potent in inducing contractlon of isolated muscle fibers of S. mansoni (T~. Day, pers. commun.) and remain the only known contractors of flatworm muscle fibers. Further characterization of these muscle-membrane prot,Jins is required if their po'Lential as drug targets is to be realized. Increased eff'ol'ts in a variety of scientific fields are needed to illuminate the physiological mer~,~ni~m~ that underlie platyhelminth neuromuscular funct!on. Direc'~ physiological characterization of the functior=s of endogenous neuromedlators i~l intact helminths and in =lispers.ed cell types as well as tJqe rr,olecular characterization and expression of the native channel and receptci proteins, will broaden our unc~erstamdin~ of these control systems. "]'he advancement of these goals ¢,'ill only be realized through the t,vpes of multidisciplinary approach that have been adopted at Michigan S~ate and Flodda State universkies.
Nematode Nervous Systems Current understanding of neuromuscular function in nematodes is more advanced than that of platyhelminths.A.O.W. Stretton (University, of Wisconsin, USA) outlined some of the data ge~erated in his laboratory through the many years of detailed experimentation required to devise a model of coordinated movement in the pig gastrointest:nal nematode. Ascar/,'ssuum. Furthermore, even with a basicunderstanding of the ar::hitecture
AIl~ghts re.served 0169-47SB/97/$17.C0 S01~.947S8(97;01034-X
|63
News of the nematode nervous ~ystem and of the fiJnctions of their classical neurotransmitters, the influence th.at endogenous neuropeptides have on neuromuscular function will greatly complicate ma~ters The number of identified nematode neuropeptides is increasing (alarmingly!) through the efforts of the Caenorhabditis elegans genome sequencing teams and the Chris Li group (Boston Universe,, USA). T.~e Stretton laboratol-/has embarked c~ a research programme to asce,~in ~ ~u,lc[ions of identified A. suum neuropeptides at the cellular level, His group has identified at least 20 peptides that might be called FaRPs (since identifying the first one nearly ten years ago), and has begun the physiological characterization of all of th~n. This work ilas been complemented by experimenLsaimed at understanding the transductlon pathways involved in FaRPactions on nematodes, carried out in the USA at Phan~acia and Upjohn4 (T.G, Geary, A.G, Maule, j.W. Bowman and O.P. Thompson) and in the UK at Southampton Unlversity s (R.J. Walker and I_ Holden-Dye). Nitnc oxide (NO), c~MP, Ca2+, K* and Clchannels have been implicated as signaling mechanisms for FaRPe~ic systems in A suum. Nematode FaRPs are aJso implicated in pharyngeal function in /~ suum fD.JA. Brownlee, Southampton University, UK) and were found to be active on trematode motility (N.J. Plarks, The Queen's University of Belfast, Uk3, indicating at least some inter-phyla conservation of FaRP-receptor structures. Peptidergic systems of nematodes pro-. vide anthelmintic targets which invite screen development and the implementation of drug-design prog~mmes. However, our knowledge of nematode neuropeptidergic systems is still extremely limited and should be expanded through concerted research efforts on numerous fronts. "Fne potential of the neuropeptidergic system for dru~ discovery, is not restri~ed to the signaling mechanisms (neuropeptides and receptors), but indudes re!.=~ed regu!atory systems, For example, neuropep'dde degradation and neurotransmltter uptake/transporter systems offer further opportunities for exploitation (P~E. Isaac, University of Leeds, UK). Althou&h current understanding of these systems in parasitic nematodes is limited, there are now numerous examples of cloned nematc,de transporters and neuropeptid~es from C. elegans, Rapid progress in this field could be made by transgenic manipulation of C. ei~oo-,r;s aimed ~t generatJng neu~transmitter-, receptor164
and tram,porter-gene deletion mutants and characterizing their phenotypes.
Ionotropic Channels The membrane-level electrophysiological effects of known nematlcides were described by R.J.Mar:in (University of Edinburgh, UK), working on A. suum. An exciting development has been the recent adaptation of the Kostyuk method to the ,'cco~ing of voltagedependent cunents in isolated muscle bag-cell prepal~tions. This technique enables more precise measuremehLs of voltage-activated currents and the intrace!lular application of test compounds during voltage-clamp procedures, The Kostyuk method has been used to identify a voltage-activated, ACh-sensitive, inward non-sc;~ctive cation current on the bag region of A. suum rnus,:le cells, This current is reduced by the FaRP, PFI. Application of this technique will likely advance our understanding of membrane level events in nematode neuromusculature and provide evidence for mechanisms of drug action. H. Wilkinson (M~rck Research Laboratories, New Jersey. USA) summarized the current understanding of the mode of action of the most successful class of antheimindc to date, the avermectins. Expression cloning studies on C elegans carded out at the Merck Research Laboratories have identified glutamate-activated CI- channels as important targets for the avermectin Jass of drugs, although their endogenous functions remain unknown 6. The importance of these channels is highlighted by the fact that similar channels have not been identified in mammals. Data obtained with C elegans knocko~¢ mutants indicate that a family of these channels occurs in nematodes, which may have multiple functions. Binding of ivermectin to C. elegans is reduced but not eliminated in these knock-out mutants (which have no detectab!e phenotype), su~esting that additional avermectin receptors are present~ N.S. Delaney and colleagues (Bath University, UI0 reported that receptor subunits, which are homolo~,.ous to some of the C elegans subunits, have been cloned from Heemonchus contortus and T.M. Skinl!er (also at Bath) raised antisera to a,~ inhib¢ory amino acid receptor subunit and localized immunos*,~ining to the lateral nerve cords of H. contortus and muscle bags of A. suum. On a cautionan/ note, I ~ Murray and R.J. Martin (University of Edinburgh, UK) reporleu "
that there may be differences between the avermectin receptors OfA. suum and C. elegans pharyngeal muscles, based on the fact that cloned C elegans glutamategaled Ct- channels are insensitive to L.-aspa~a;e, whereas it increases the input con&,c'tance of A. suum pharyngeal muscles. Other nematode-muscle channel proteins have been implicated as sites of action for nematicidal drugs, including levamisole. This drug acts on nicotinic acety'~choline receptors ,'nAChRs), which occur as members of a multigene family in nematodes (D.B. S~elle, University of Cambrid£e, USA) Molecular cloning methods (card_,d out at Cambridge University, Mass'~chusetts General Hospital Cancer Center and the University, of Texas at S~JnAntenio) have identified a !arge Family of C. elegens AChR subunits w:~ich are closely related to analogous i~.sect proteins. !mpor~.~ntly, ,-elated subunits have been cloned "From th~ parasitic forms, TrichostronEylus colubrifon'nis and Onchocerca vohulus. Although levamisolesensitive currents can be detected in Xenopus oocytes after the injectie~ of cRNAs encoding C. elegansnAChR subunits, expression is not robust and it is not known if these are identical to the wild-type receptor. In comparison with mammalian nAChRs, the nematode analogues display some unique pharmacological characteristics, such as their insensitivity to lophotoxin, which can be understood on the molecular level. Such chara~cristics suggest that future exploitation of parasitic nematode ionotropic receptors may be profitable for anthelmintic discovery.
Anthelmintic Resistance A most worrisome development for parasite control is the ever-increasing catalogue of drug-resistant strains. Resistance has been reported for several of the drugs which specifical',ytarget (or are believed to target) parasitic helminth neuromuscular systems including levamisole, ivermectin and praziquantel. Current knowledge of the pharmacology of anthelmintic resistance in heh,',ii,th pa~s~es was summarized by N. Sangster (University of Sydney, Australia). it is clear that detailed understanding of the mechanisms of anthelmintic resistance will shed light on how these drugs work and on the biology of the systems they target. Molecular genetic studies on C eleganshave greatly advanced our understanding of benzimidazole-resistance and are likely to be critical in further characterization of Paras:'.oloEyTc~y. vs!. !3. he. 5. !oq7
N ews resistance to neuro- and myo-actlve compounds. In th;s way, P. Hunt (University of Edinbupgh. UK) and col~eagues screened a large number of iverrnectinresistant C, elegans to identify genes which confer and suppress iverrne~in ~sistance. Un!!k~ the case in 9ara~if,_ nemato:':es, ivermectin resistance in C e/ego ~ is typically recessive. The new gen~s are at least semi-dominant, wn;ch make them of more relevance for he situation in parasites. They also pn=sented evidence that suggested that iverme~in resistance is linked to am!:}h~d-deHved pharyngeal modulation. The prospects for rational api~roaches to anthelmintlc discovery were discussed b T T,G. Gear-/(Pharm ,c;a and Upjohn Co,, USA). The concept of rational drug discover,/ was introducec ~ in the cor.text that other forms of drug discover/must, by analogy, be 'irrational', wh~cq is certainly an irrational conclusion. P.andom s~reening oroglamrnes hace bee;; funclamental in the discover,/ of all current front-line anthelmintics. Ne~er developments in the pharmaceutical industry have advanced high-throughput random screening in mechan;sm-based assays (as opposed to who!e organism toxici b' assays) to be often favoured over computationally based drug design strategies. Anthelmintic discovery programmes can adopt this same strateE/ if we gain greater understanding of the biology of these organisms. Once good
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drug targets are identified, they car, be inct ~oorated into mechanism-based screens. Achieving this understanding is absolutely dependent upon increased basic research on nematode hieing', indudir, f, neurobiology. Again, the irr,pr~tanr~ ~" the C. ele~ans £ont~m~ sequencing project to the identification of new anthelmintic ta~gets A,as emphasized.
e~.cellcn~ opportunity to form a coherent community of iike-minded sc!entists. Based on its success, plans a_-ebein~ made for a similar gathering in 1998, On behalf of the delegate'~ we wouid like to thank the organizers, ,~o¢. De,,;4 'vV Halton (The Queen'.; Unive,sity of 3elfast) and Dr P.!c%=~'. Me-tin (ErJinburgh ;Jniversity) for a highly successfvl meeting. The event was very generously sponsored by Bayer, BSP, Janssen Anima! Health, Pfizer Central Research, Pharmacia and Upjohn, The Wellcome Trust and Zeneca. All 13 of O n a Lighter N o t e the plcnar,/lectures are due to be published The conference was concluded by a in a special supplement of Parasitology '~. highly convivial Chinese banquet which was thoroughly enjoyed by all invited References I }ohr,ston.R.N. e[ al. (1996)J. Neurochem.67, sF.eakers and sessio~ chairs (except R.]. 814-821 2 Day, TA. et el. (1994) Parosito!e~y i09. 'ffartin to whom the bill was pre455-459 ~ented!). The best pos[er presentation 3 Marks. N.J. et at. (,1996) Parasitology 113. won a bottle of whisky, which went to 393-40~ Gunnar Mair (The Queen's University 4 l'4aule.,~-G.et eL (1996.1PorositoL Toda,~ 12. of Belfast. UI0 for his study of #laW35t.-357 5 Brownlee.D.J~ et aL (:996) Pcr,Tsitol. Todct;, helminth musculature using fluore.-'t~_r,'~ 12. 343-35I tags and confocal microscopy. 6 I:tter. A. et ell (t'~q~ J. 6ioL Chem. 271, 16035-16O39 Acknowledgements 7 Halton. DW. ('99) Paras;toto~/ 102, On 18-19 September 1996. a special symSLS118 8 Ha,~nn, D.W. (1996)Parositology I 13,51-5238 posium entitled 'The molecular biochemistry and ph~iolo~,;, ~f helminth neuromuscula,- systems' was held at C~.," Univer~,itv. A G ~ a I~,aule is at The Comparative NeuroLondon. This was the second , .ch s'~mF.o- endocrinology Pesearch Group, Schools of 8ioiogy and Biooh6mist~ and Clinical Medislum and followed the very successful ~bsh Society for Par~s~toloK)' (B~.;P) meet:,'b on cine, ,~heQueen's University of Belfast, ~elfast, Pa~-asite Neurobiology which was heL5 at UK BT7 JNN. Tim Gear), is at/,n,mal Health the same venue7. The sym/.,osium was w2:- Discover, Research, Pharmacia and Upjohn refuted by the recent surge ,n the numb,:r Co., Kaiamazoo, MI 49001, USA. Tel: . t ~ of laboratories working on h~Iminth ne,Jro- 12J2 272059, lax'. +44 1252 256S05, biolo.~ayon ~o,pl~i~:icatedlevels, ar# was an e-n',~/: a.nwu'~,~,_,~.a-_ uk
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