Unravelling the moulting degradome:new opportunities for chemotherapy?Hannah Craig1, R. Elwyn Isaac1 and Darren R. Brooks2

1 Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds, West Yorkshire,

LS2 9JT, UK2 Biomedical Sciences Research Institute, School of Environment and Life Sciences, University of Salford, Salford, Greater

Manchester, M5 4WT, UK

Opinion TRENDS in Parasitology Vol.23 No.6

Glossary

Apolysis: the separation of the cuticle from the epidermis in members of the

ecdysozoa.

Collagen: long fibrous structural protein composed predominantly of glycine-

X-Y triplet amino acid repeats.

Degradome: the complete repertoire of peptidases in an organism or the

subset of peptidases that is involved in a defined biological event.

Ecdysozoa: group of animals that possess a cuticle that is periodically shed

during development, includes arthropods and nematodes.

Ecdysis: periodic shedding of the exoskeleton or cuticle.

Hypodermis: single layer of cells that underlies and is responsible for secreting

the nematode cuticle.

Proteasome: complex intracellular structure that is responsible for protein

degradation and turnover.

RNA interference (RNAi): a mechanism whereby fragments of double-stranded

Replacement of the nematode cuticle with a newlysynthesized cuticle (a process known as moulting)occurs four times during larval development. Therefore,the key components of this essential developmentalprocess represent attractive targets for new chemothera-peutic strategies. Recent advances in understandingthe molecular genetics of nematode moulting shouldstimulate and facilitate development of novel drugs thattarget the essential molecules of the moulting cycle. Inparticular, we argue that further understanding of themoulting degradome and its key peptidase membersoffers an important opportunity for the development ofnovel antinematode agents.

Targeting the moultThe ecdysozoa (see Glossary) surround their bodies with acuticle that supports body shape, provides a protectivebarrier against the external environment and functions asan exoskeleton for the anchorage of locomotory muscle.The rapid growth of the juvenile stages of ecdysozoameansthat the exoskeleton can be quickly outgrown; therefore,these animals must periodically synthesize and lay downa new larger cuticle to enable the old confining cuticle tobe shed. This moulting cycle is a complex sequence ofevents that involves changes in metabolism and beha-viour, and can result in complete or partial metamorpho-sis. During a moult, the new cuticle must be protectedwhile the old one is degraded and cast off, and linksbetween muscles and the exoskeleton have to be detachedand reformed.

This elaborate series of events presents severalopportunities for the development of strategies to disruptgrowth and development in pest species. Indeed, twoclasses of chemicals are established insect-growth regula-tors and are used in the field to control insect populations.The acylureas target the moulting cycle by disrupting thesynthesis of chitin, a major component of the insect integu-ment, and the bisacylhydrazines mimic the action ofthe steroidal moulting hormone (20-hydroxyecdysone) tocause inappropriate and lethal moults [1,2]. An attractivefeature of these chemicals is their specificity and non-neurotoxic mode of action.

Corresponding author: Brooks, D.R. (d.r.brooks@salford.ac.uk).Available online 24 April 2007.

www.sciencedirect.com 1471-4922/$ – see front matter � 2007 Elsevier Ltd. All rights reserve

Recent advances in understanding nematodemoultingThe nematode cuticle is a complex, three-layered structurethat is composed predominantly of collagen [3]. Duringeach larval stage, the nematode enters a period of inactiv-ity, known as lethargus, when the old cuticle begins todisconnect from the underlying hypodermis. Apolysis ofthe old cuticle subsequently occurs and this enables thenewly synthesized cuticle to be secreted into the space thathas been created. After movement to loosen the old cuticle,ecdysis occurs [4,5].

Several important criteria make targeting the moultingcycle of parasitic nematodes an attractive strategy for thedevelopment of new nematocides –moulting is an essentialdevelopmental pathway, it is likely to be well conservedacross the phylum and it is absent from host species(vertebrates and plants). Recent research, primarily usingthe free-living nematode Caenorhabditis elegans [6–8](Table 1), has provided new molecular insights into themechanism of moulting in nematodes and has identifiedpotential targets for chemotherapy of parasitic-nematodeinfections. Among this set of moult genes, the importanceof peptidases (Box 1) is becoming increasingly apparent,not only for controlled synthesis of the new cuticle andrelease of the old but also asmediators of signalling events.Further understanding of the moulting degradome, and inparticular the roles of the essential peptidases, should offerexciting opportunities for the development of new che-motherapeutics that disrupt nematode moulting.

RNA specifically silence gene expression. The pioneering RNAi research with

Caenorhabditis elegans has revolutionised the study of gene function, as

recognized by the award of the 2006 Nobel Prize in Physiology or Medicine.

Seam cell: specialized epithelial cells that lie along the apical midline of the

hypodermis.

d. doi:10.1016/j.pt.2007.04.003

Table 1. Summary of the known peptidases and peptidase inhibitors essential for nematode moulting

Class Organism Gene or gene product Possible function Refs

Peptidases Metallo Caenorhabditis elegans DPY-31 Processing of cuticular collagen [51,52]

C. elegans NAS-36 Apolysis and/or ecdysis [6,52]

C. elegans NAS-37 Apolysis and/or ecdysis [6,52]

C. elegans ADT-2 Processing of cuticular collagen [6,8]

Serine C. elegans BLI-4 Processing of cuticular collagen [6]

C. elegans y54e10br.5 Signal peptidase [44]

Cysteine C. elegans QUA-1 Signalling in hypodermal cells [6]

Onchocerca volvulus Ov-CPL Apolysis and/or ecdysis of L3 cuticle [37]

O. volvulus Ov-CPZ

Aspartic C. elegans IMP-2 Sterol homeostasis essential for moulting [42]

Threonine C. elegans PBS-5 Catalytic subunit of the proteasome, which is

responsible for protein turnover

[8,44]

Peptidase

inhibitors

Serine C. elegans BLI-5 Regulation of enzyme involved in collagen

processing

[6,56]

C. elegans MLT-11 Regulation of ecdysis [6]

Other Nonpeptidase C. elegans ACN-1 Signalling to mediate ecdysis [6,54]

Proteasome noncatalytic

a-subunit

C. elegans PAS-1 Protein turnover [44]

C. elegans PAS-6 Protein turnover [8]

C. elegans PAS-7 Protein turnover [44]

Proteasome noncatalytic

b-subunit

C. elegans PBS-5 Protein turnover [8,44]

Proteasome activator

complex subunit

C. elegans RPN-7 Protein turnover [44]

Opinion TRENDS in Parasitology Vol.23 No.6 249

Peptidases as attractive targets for disease controlUndoubtedly one of themajor challenges is the developmentof biocides that are active against parasitic nematodes butwhich are relatively nontoxic to the host. Encouragingly,there are several excellent examples of the development ofhighlyselectivepeptidase inhibitors [9].Forexample, aspar-tic-peptidase inhibitors are used in the treatment of HIVinfection [10]. Interestingly, there is evidence that aspartic

Box 1. Peptidases

Hydrolysis of peptide bonds, termed proteolysis, is mediated by

enzymes known as proteases, proteinases or peptidases [50].

Peptidases can be divided into two major groups: the exopepti-

dases, which remove one or a small number of amino acids from

the N or C terminus of a protein or peptide, and the endopeptidases,

which hydrolyse internal peptide bonds.

Peptidases are further subdivided based upon their catalytic

mechanism.

� Aspartic peptidases – In most aspartic peptidases, a pair of

aspartic acid residues bind and activate a water molecule that

then works as a nucleophile to attack a peptide bond. In some

aspartic peptidases, an alternative second amino acid might be

involved.

� Metallopeptidases – These also employ an activated water

molecule as a nucleophile. The water is activated by a divalent

metal ion, most commonly zinc, that is held in place by amino acid

ligands. In some metallopeptidases, two metal ions are required

for catalysis.

� Cysteine peptidases – An activated sulfydryl or thiol group on a

cysteine residue works as the nucleophile. A conserved histidine

residue, which works as a proton donor, is also required for

activity.

� Serine peptidases – These have a similar mechanism to the

cysteine peptidases. A hydroxyl group of a serine residue works

as the nucleophile with either histidine or, in some cases, lysine as

the proton donor. In those with a histidine as proton donor, a third

conserved residue, either aspartate or another histidine, is

required for activity, making up what is known as a catalytic triad.

� Threonine peptidases – All belong to the N-terminal nucleophile-

hydrolase class and possess an N-terminal catalytic threonine

residue. The catalytic subunits of the proteasome are included in

this group.

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peptidasesof several opportunistic parasites that infectHIVpatients might be susceptible to HIV inhibitors [11]. Highlyselective inhibitors of the human M2 and M13 peptidasefamilies have also been successfully developed to treathypertension, arthritis, diabetes and renal disease [12–14] and several members of the M12 family are currentlyhigh-profile targets in drug discovery programmes aimed atdevelopingmore effective treatments for rheumatoidarthri-tis and tumour angiogenesis and growth [15,16].

We believe that existing drug discovery strategies thatare used in the development of these important thera-peutics can also be applied to developing a new generationof antinematode drugs that are based on inhibition of keytargets in the moulting degradome. One foreseeable chal-lenge to synthetic chemists is the impermeable nature ofthe cuticle. However, earlier studies with peptidase inhibi-tors demonstrate that metallo- and cysteine-peptidasetargets are accessible [17]. The following sections highlightsome of the key peptidases that are potential targets forlead-compound development.

Metallopeptidases

AHaemonchus contortus zinc metallopeptidase is secretedinto the exsheathing fluid and is likely to be involved increation of the refractile ring that results in release of theanterior tip of the larval stage 2 (L2) cuticle, which enablesthe infective L3 to escape [18]. Application of metallopep-tidase-specific inhibitors to in vitro cultures of Brugiapahangi and Ascaris suum has demonstrated the require-ment for metallo-aminopeptidase activity during the L3 toL4 moult [19–21]. In another filarial nematode, Dirofilariaimmitis, metallopeptidase activity is elevated in themoult-ing L3 [22].

Key metallopeptidases that are involved in C. elegansmoulting are described in detail in Box 2. Encouragingly,homologues of C. elegans moult metallopeptidases arepresent in parasitic-nematode databases. For example,BLAST searches have identified predicted protein

Box 2. Metallopeptidases and moulting in Caenorhabditis elegans

The C. elegans proteins NAS-36, NAS-37, DPY-31, ADT-2 and ACN-1

have key roles in cuticle synthesis and replacement [51–54] (Figure I).

The first four are members of the astacin–adamalysin M12 family and

are predicted to be secreted peptidases with multiple functional

protein domains [55]. nas-36 and nas-37 gene expression is mainly

restricted to hypodermal cells of L1 to L3 larvae and the L4 seam cells

when they terminally fuse with hyp-7. NAS-37 is more likely to be

important for apolysis and ecdysis per se, rather than the synthesis

and maturation of new cuticle [53].

DPY-31 is strongly expressed in embryos and early larvae and

might serve as a procollagen C-peptidase that is involved in the

maturation of cuticular collagen (including SQT-3 collagen) by the

removal of carboxyl propeptides from procollagen [51]. dpy-31

mutants display several cuticular defects that result in a highly

penetrant embryonic-lethal phenotype [51].

ADT-2 is related to mammalian tumour necrosis factor (TNF)-a

converting enzyme (TACE), the enzyme that is responsible for the

shedding of TNF-a and other cell-surface proteins [13] and is also

related to mammalian procollagen N-endopeptidase, which is

responsible for the N-terminal processing of procollagen I and II

[50]. Like NAS-36 and NAS-37, ADT-2 has a peptidase domain that

includes a predicted proprotein region. The peptidase domain is

followed by a single thrombospondin (TSP)-1 domain, a cysteine-rich

region and then six TSP-1 domains. RNAi gives rise to several

phenotypes, including abnormal locomotion, a dumpy phenotype

and larval arrest because of a failure to moult properly [8].

ACN-1 is an unusual member of the angiotensin-converting enzyme

(ACE) family of metallopeptidases and has several protein domains

that are thought to be involved in determining protein localization and

protein–protein interactions [54]. C. elegans ACN-1 has a similar

expression pattern to that of NAS-37 and is also required for the

shedding of old cuticle (see Figure 1 in the main text). acn-1

expression in the hypodermal cells of C. elegans is regulated by

nuclear hormone-receptor genes and controls expression of down-

stream genes in the genetic pathway that regulates nematode ecdysis

[6,54].

Figure I. Schematic of the protein domain structures of metallopeptidases involved in Caenorhabditis elegans moulting. NAS-36, NAS-37, DPY-31 and ADT-2 possess a

number of common protein domains. These include: a signal peptide (SP) secretory sequence for transit to the cell surface; a proprotein domain (dark blue banner),

removal of which leads to peptidase activation; a peptidase domain (yellow); epidermal growth factor (EGF)-like domain (pink); CUB domain (grey); an extracellular

domain that is present in a wide range of proteins, including developmentally regulated peptidases, which contains four conserved cysteine residues that probably form

two disulphide bridges; thrombospondin (TSP)-1-like domain (brown) and a cysteine (CYS)-rich domain (green). In addition to the SP sequence and a cysteine-rich

domain, ACN-1 has a proline–glutamate-rich region (P–E, red), O-glycosylation (mucin-like) sites (O-G, white) and a predicted GPI-anchor attachment site (black).

250 Opinion TRENDS in Parasitology Vol.23 No.6

sequences from Trichinella spiralis and Meloidogynechitwoodi that share 72% and 52% identity, respectively,with DPY-31 (H. Craig et al., unpublished) (see http://www.wormbase.org for gene and protein name definitions).Sequences that share considerable identity with ACN-1

Figure 1. Disruption of Caenorhabditis elegans moulting by RNAi. Transmission electron

and (b) an arrested L4 larva trapped inside the L3 cuticle (outer ring). The new cuticle

between the two cuticles as a consequence of some partial shedding. Figure (b) was o

stranded RNA.

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are present in T. spiralis and a range of other nematodes,includingMeloidogyne incognita, B. malayi and Parastron-gyloides trichosuri (H. Craig et al., unpublished), indicatingthat this ‘nonpeptidase’ has a conserved role in nematodemoulting (Figure 1).

microscopy images of transverse sections through (a) a wild-type adult C. elegans

(inner ring) and struts are disorganized and Escherichia coli bacteria are trapped

btained by feeding L1 and L2 nematodes with E. coli that expressed acn-1 double-

Opinion TRENDS in Parasitology Vol.23 No.6 251

Serine peptidases

Recent data have indicated that serine peptidases andtheir endogenous inhibitors (Box 3) are crucial regulatorsof nematode moulting. The bli-4 locus ofC. elegans encodesat least nine subtilisin-like proprotein convertases byalternative splicing of 12 common exons to specific down-stream sequences [23]. Mutations that affect certain iso-forms of BLI-4 are associated with blistering of the adultC. elegans cuticle and, hence, bli-4 has been attributed afunction in moulting. More specifically, BLI-4 seems to beinvolved in the processing of cuticular collagens duringnew cuticle formation. C. elegans cuticular collagens con-tain a proprotein convertase-like R-X-X-R cleavage motifwithin the highly conserved homology block A (HBA) andthere is evidence that cleavage at this site is essential forcorrect cuticle assembly [24,25]. Further evidence comesfrom the discovery thatmutations of the dpy-5 collagen cansuppress bli-4, indicating that the presence of abnormalDPY-5 collagen might somehow counteract blistering thatis caused by collagens that are substrates of BLI-4 [26].

A blisterase gene with a predicted catalytic domain thatshares 84% identity with bli-4 has been characterized inthe filarial nematode Onchocerca volvulus [27]. BLASTsearches of available expressed sequence tag (EST) datasetshave identified cDNAs with predicted protein sequencesthat share considerable identity with BLI-4 from severalother parasites of animals, such as T. spiralis and H. con-tortus, and parasites of plants, such as M. chitwoodi andXiphinema index (H. Craig et al., unpublished). Because nodataonconservationofHBAs inO.volvulus cuticle collagensare currentlyavailable, apossible role in collagenprocessing

Box 3. Endogenous serine-peptidase inhibitors

Specific endogenous peptidase inhibitors are likely to be important

during nematode moulting by regulating the activities of degrado-

mal components. The genome-wide RNAi screen for moulting

phenotypes in Caenorhabditis elegans identified genes that encode

predicted serine-peptidase inhibitors (SPIs) [6]. One of these, bli-5,

has been identified in an RNAi screen for genes involved in cuticular

collagen biosynthesis [56]. BLI-5 is a secreted kunitz-type SPI that is

highly expressed in the larval and adult hypodermis and bli-5 RNAi

results in blister and moult defects and disruption of COL-19

collagen [56]. The genomic sequence of mlt-11 predicts ten kunitz-

type SPI-like domains. mlt-11 is transiently expressed in the

hypodermis and seam cells before each moult and mlt-11 RNAi

results in failure to shed the old larval cuticle during moulting [6].

It is thought that BLI-5 might interact with BLI-4 because their

RNAi phenotypes and tissue distribution are comparable or it could

regulate the activity of another functionally similar enzyme [56].

Other enzymes involved in collagen processing and ecdysis, such as

DPY-31 and NAS-37, might require cleavage at an R-X-X-R site for

activation [51,53,56]. Inhibitors such as BLI-5 and MLT-11 could,

therefore, have an important role in cuticle biosynthesis and

moulting by inhibiting collagen-processing enzymes or by regulat-

ing subtilisin-like processing of such enzymes. Kunitz-type SPIs are

present in other nematode species and could have potential as

protective antigens against Ancylostoma caninum and Anisakis

simplex, although confirmation of a role in moulting is awaited

[57,58].

Moulting in Onchocerca volvulus has been shown to require SPIs

that possess trypsin-inhibitor-like domains. Ov-SPI localizes to the

new cuticle and between the old and new cuticles during L3

moulting and Ov-SPI RNAi results in failure to shed the old cuticle

[59]. SPIs have been identified in numerous other nematode

species, although further functional characterization is required.

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is unconfirmed.However, cuticular collagen genes thathavebeen identified in other nematode species, includingH. contortus, Meloidogyne javanica and M. incognita, dopossess the R-X-X-R cleavage motif [28–30]. Vertebrateprocessing of procollagen is characteristically performedby metallopeptidases [31,32]. This evidence, however,points towards a unique involvement for subtilisin-likeenzymes in procollagen processing that has been conservedin nematodes. If this is the case, then it might be possible todevelop a blisterase-specific nematocide.

Cysteine peptidases

The application of cysteine-peptidase inhibitors to larvalcultures of several species of parasitic nematode hasdemonstrated the requirement for this class of peptidasein moulting [17,21,33–35]. Furthermore, the precise regu-lation of cysteine-peptidase activity is likely to be crucialduring the moult, as indicated by the presence of naturalcysteine-peptidase inhibitors, the cystatins, in parasiticnematodes [36]. Recent work using RNA interference(RNAi) has identified O. volvulus cathepsin L and Z-likecysteine peptidases as essential components of the L3moult [37]. The expression pattern of the C. elegans cpl-1gene is also indicative of a probable role in moulting [38].

In C. elegans, the cysteine peptidase that is encoded byqua-1 has been shown to be an important mediator of earlysignalling events in the hypodermal cells [6]. QUA-1 is anematode-specific member of the hedgehog signallingfamily and comprises an N-terminal Qua domain and aC-terminal Hint–Hog catalytic domain that is responsiblefor an unusual autocatalytic cleavage [39,40]. The target ofthe secreted Qua signalling domain is unknown and couldinclude members of the expanded family of patched andpatched-related membrane receptor proteins, in additionto several other predicted signalling pathway components[41]. Homologues of qua-1 have been identified in othernematodes, including filaria and parasitic nematodes ofplants; therefore, QUA-1 represents a potential target forchemotherapeutic development.

Aspartic peptidases

The imp-2 gene of C. elegans is distantly related to thepresenilin family of intramembrane peptidases. Geneknockdown by RNAi demonstrates that imp-2 is essentialfor the completion of embryogenesis and moulting [42].BLAST searches have revealed predicted proteinsequences that share a high level of identity with IMP-2from parasitic nematodes such as Necator americanus,Globodera rostochiensis, Strongyloides stercoralis and Tri-churis muris (H. Craig et al., unpublished). Given thatpresenilins are drug targets for Alzheimer’s disease [43],the potential exists for screening large chemical librariesfor inhibitors with activity against IMP-2 homologues ofparasitic nematodes.

Other peptidases

The mechanics of moulting places particular emphasis onpeptidases that are required for cellular trafficking andprotein turnover. An RNAi screen in C. elegans [44] indi-cated that the signal peptidase that is encoded by thegene y54e10br.5 is essential for moulting. Targeting the

252 Opinion TRENDS in Parasitology Vol.23 No.6

C. elegans proteasome subunits by RNAi has indicated thatseveral noncatalytic a-subunits (pas-1, pas-6 and pas-7),one catalytic b-subunit (pbs-5) and a subunit of the protea-some activator complex (rpn-7) are essential for moulting[8,44]. Proteasome inhibitors such as the peptide alde-hydes lactacystin and gliotoxin, although not totallyspecific, do elicit effects on parasites [45]. In addition,bortezomib (PS-341), the selective dipeptide boronate,has been approved for clinical use in treatment of multiplemyeloma [46]. Such compounds might be candidates toassess as potential nematocides.

Concluding remarksGiven the ancestral roots of moulting, many targets arelikely to be well conserved among nematodes and, hence,provide good opportunities for the development of newantinematode agents. Successful replacement of the nema-tode cuticle is dependent upon being able to lay down newcuticle while at the same time detaching the old cuticle.The secretion of peptidases within the moult cycle to elicitdetachment is seen as a key event while, at the same time,newly synthesized cuticle must be protected from proteo-lytic attack. We envisage fine control of proteolysisthroughout the moult through timing of peptidase expres-sion, enzyme activation and expression of peptidase inhibi-tors.

Reverse genetic analyses in C. elegans have shown thatthe moulting degradome includes peptidases from allknown major classes, including key roles for several mem-bers and also peptidase inhibitors. In addition to continu-ing to exploit the genetic manipulability of C. elegans,continued development of robust RNAi tools in parasiticnematodes [47,48] is crucial to dissecting the roles of keydegradomal components further. Peptidases are oftenfound within multigene families that tend towards func-tional redundancy and, therefore, a re-evaluation of theuse of combinatorial RNAi is also required [49].

Further understanding of the roles of peptidases inmoulting should enable target prioritisation in parasiticnematodes and provide opportunities to develop new leadcompounds. To facilitate drug development, C. eleganscould be used as the target organism to screen largechemical banks of peptidase inhibitors and establish whichcompounds are capable of crossing the cuticle. Becausecuticle composition varies among species and stages ofthe life cycle [5], potentially efficacious compounds shouldbe rapidly tested for activity towards parasitic nematodeswith amenable life-cycle cultivation. Given the widespreadappearance of resistance to established anthelmintics andthe threat that this poses to human and animal health,new lead compounds with potency and selectivity againstnovel targets are now overdue.

AcknowledgementsWe acknowledge Clare Nicol and Adrian Hick for their electronmicroscopy work and the Biotechnology and Biological SciencesResearch Council for funding (BBS/B/11192).

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Free journals for dev

The WHO and six medical journal publishers hav

Research Initiative, which enables nearly 70 of the

to biomedical literature

The science publishers, Blackwell, Elsevier, Harc

International Health and Science, Springer-Verlag a

and the British Medical Journal in 2001. Initially, m

free or at significantly reduced prices to universit

institutions in developing countries. In 2002, 22 ad

journals are now available. Currently more than 7

Gro Harlem Brundtland, the former director-gen

‘‘perhaps the biggest step ever taken towards red

and poor co

For more information, vis

www.sciencedirect.com

45 Paugam, A. et al. (2003) Characterization and role of protozoanparasite proteasomes. Trends Parasitol. 19, 55–59

46 Richardson, P.G. et al. (2005) Bortezomib or high-dose dexamethasonefor relapsed multiple myeloma. N. Engl. J. Med. 352, 2487–2498

47 Zawadzki, J.L. et al. (2006) RNAi inHaemonchus contortus: a potentialmethod for target validation. Trends Parasitol. 22, 495–499

48 Knox, D.P. et al. (2007) RNA interference in parasitic nematodes ofanimals: a reality check? Trends Parasitol. 23, 105–107

49 Geldhof, P. et al. (2006) Combinatorial RNAi on intestinal cathepsin B-like proteinases in Caenorhabditis elegans questions the perceptionof their role in nematode biology. Mol. Biochem. Parasitol. 145, 128–132

50 Barrett, A.J. et al. (2004) Handbook of Proteolytic Enzymes. ElsevierAcademic Press

51 Novelli, J. et al. (2004) Gene interactions in Caenorhabditis elegansdefine DPY-31 as a candidate procollagen C-proteinase and SQT-3/ROL-4 as its predicted major target. Genetics 168, 1259–1273

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53 Davis,M.W. et al. (2004) A conservedmetalloproteasemediates ecdysisin Caenorhabditis elegans. Development 131, 6001–6008

54 Brooks, D.R. et al. (2003) An essential role in molting andmorphogenesis of Caenorhabditis elegans for ACN-1, a novelmember of the angiotensin-converting enzyme family that lacks ametallopeptidase active site. J. Biol. Chem. 278, 52340–52346

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57 Chu, D. et al. (2004) Molecular characterization of Ancylostomaceylanicum kunitz-type serine protease inhibitor: evidence for a rolein hookworm-associated growth delay. Infect. Immun. 72, 2214–2221

58 Shimakura, K. et al. (2004) Purification and molecular cloning of amajor allergen from Anisakis simplex. Mol. Biochem. Parasitol. 135,69–75

59 Ford, L. et al. (2005) Characterization of a novel filarial serine proteaseinhibitor, Ov-SPI-1, from Onchocerca volvulus, with potentialmultifunctional roles during development of the parasite. J. Biol.Chem. 280, 40845–40856

eloping countries

e launched the Health InterNetwork Access to

world’s poorest countries to gain free access

through the internet.

ourt Worldwide STM group, Wolters Kluwer

nd John Wiley, were approached by the WHO

ore than 1500 journals were made available for

ies, medical schools, and research and public

ditional publishers joined, and more than 2000

0 publishers are participating in the program.

eral of the WHO, said that this initiative was

ucing the health information gap between rich

untries’’.

it www.who.int/hinari