P A R A S I T E B I O L O G Y A N D B I O C H E M I S T R Y
H o w e v e r , t w o cuticlin g e n e s , cut-l a n d cut-2, h a v e n o w b e e n isolated from C. elegans in this laboratory, using a D.
melanogaster p r o b e c o d i n g for a c o m p o n e n t o f t h e vitelline m e m b r a n e o f the egg. cut-l mRNA is 1 4 2 2 nt long, h a s four e x o n s c o d i n g for 4 2 3 a m i n o acids a n d is transpliced to SL1, t h e s p l i c e d l e a d e r p r e s e n t at the 5' e n d o f m a n y mRNA's in m o s t n e m a t o d e s ; cut-2 mRNA is 8 4 7 nt long, c o n t a i n s o n l y t w o e x o n s c o d i n g for 2 3 7 a m i n o acids, a n d is n o t transpli
ced. Northern analysis indicates that w h i l e cut-l is transcri
b e d stage-specifically b y w o r m s entering the d a u e r larvae stage, cut-2 mRNA is t r a n s c r i b e d during cuticle synthesis, immediatly b e f o r e e a c h moult.
Parts o f b o t h g e n e s h a v e b e e n e x p r e s s e d as fusion proteins in E. coli and h a v e b e e n u s e d to raise s p e c i f i c a n t i b o d i e s . T h e s e h a v e b e e n u s e d to study the e x p r e s s i o n pattern of the t w o g e n e s b y w e s t e r n blot, a n d to l o c a l i z e the p r o d u c t s within the c u t i c l e s o f w o r m s at different s t a g e s b y i m m u n o f l u o r e s c e n c e a n d i m m u n o - e l e c t r o n m i c r o s c o p y . T h e results o b t a i n e d confirm the partial s t a g e specifity o f C U T - 1 , a n d t h e fact that C U T - 2 is a c o m p o n e n t o f t h e c u t i c l e at all stages. B o t h proteins are l o c a l i z e d o n cuticle residues after treatment with strong reducing a g e n t s , s h o w i n g t h e m to b e definitively m e m b e r s o f t h e cuticlin residue.
T h e proteins deriving from the c o n c e p t u a l translation o f the g e n e s differ substantially, a l t h o u g h t h e y b o t h b e g i n with signal p e p t i d e s a n d s h a r e a short motif r e p e a t e d 5 t i m e s in CUT-1 a n d 12 t i m e s in C U T - 2 . E a c h repetition is characteri
z e d b y t h e p r e s e n c e o f t h e a m i n o a c i d s e q u e n c e AAPA.
T h i s s a m e m o t i f c a n also b e found in s o m e o f the vitelline m e m b r a n e p r o t e i n s o f Drosophila a n d in t h e p r o t e i n s w h i c h m a k e up the c u t i c l e o f Locusta migratoria. T h e s e p r o t e i n s are all i n v o l v e d in the formation o f i n s o l u b l e , p r o t e c t i v e e x t r a c e l l u l a r l a y e r s , i m p l y i n g that t h e c o n s e r v e d d o m a i n s m a y h a v e an important functional role.
I n t e r e s t i n g l y , t h e a m i n o a c i d s e q u e n c e o f C U T - 2 s h o w s tyrosine residues that c o u l d participate in dityrosine bridge formation (dityrosine is p r e s e n t in the i n s o l u b l e residue o f parasitic n e m a t o d e c u t i c l e s ) . W e h a v e s h o w n that s o l u b l e r e c o m b i n a n t C U T - 2 , p r o d u c e d in E. coli, c a n b e p o l y m e r i z e d in vitro i n t o h i g h m o l e c u l a r w e i g h t s p e c i e s b y t h e action o f HR p e r o x i d a s e in the p r e s e n c e o f H 2 O 2 . T h e pro
d u c t s o f the r e a c t i o n b e c o m e i n s o l u b l e , c o n t a i n t y r o s i n e a n d the r e a c t i o n is inhibited b y the p r e s e n c e o f free tyro
sine. T h i s clearly b e g s the q u e s t i o n w h e t h e r CUT-2 is res
p o n s i b l e for the insolubility o f the cuticle.
A s e c o n d g e n e s h o w i n g significant h o m o l o g y ( > 8 0 % ) to cut-l h a s b e e n isolated for C. elegans, confirming the p o s sible e x i s t e n c e o f a cuticlin g e n e family. A cut-l h o m o l o g u e h a s a l s o b e e n isolated from the plant parasitic n e m a t o d e , Meloidogyne artiella, d e m o n s t r a t i n g the strongly c o n s e r v e d nature o f the s e q u e n c e a m o n g s t n e m a t o d e s .
An i n s o l u b l e cuticlin residue is p r e s e n t in the c u t i c l e s o f all n e m a t o d e s s t u d i e d s o far. T h i s fact, p l u s t h e a p p a r e n t l y c o n s e r v e d nature o f the g e n e and the protein for w h i c h it c o d e s , h a s p r o m p t e d t h e s e a r c h for g e n e s h o m o l o g o u s to cut-l a n d cut-2 in t w o parasitic n e m a t o d e s , Ascaris lumbri- coides and Brugiapahangi.
T w o parallel a p p r o a c h e s h a v e b e e n u s e d : the first involves s c r e e n i n g a g e n o m i c l i b r a r y w i t h l a b e l l e d D N A p r o b e s m a d e from C. elegans cuticlin g e n e s ; the s e c o n d involves s c r e e n i n g a parasite c D N A e x p r e s s i o n library with the s p e
cific a n t i b o d i e s raised against the r e c o m b i n a n t C U T - 1 a n d C U T - 2 purified p r o t e i n s . T h e p o s i t i v e c l o n e s h a v e b e e n s u b - c l o n e d into the p B l u e s c r i p t p h a g e m i d s y s t e m a n d are at p r e s e n t b e i n g s e q u e n c e d . T h e s e q u e n c e s will b e c h e c k e d for h o m o l o g y against the C. elegans cuticlin g e n e s a n d the S e q u e n c e s Data B a s e .
O n c e c u t i c l i n g e n e h o m o l o g u e s h a v e b e e n f o u n d t h e a p p r o a c h to their study will f o l l o w t w o b r o a d p a t h w a y s . Firstly transcription patterns in the parasite life-cycles will b e s t u d i e d u s i n g r e v e r s e t r a n s c r i p t a s e a n d P C R . A n d s e c o n d l y t h e inserts will b e e x p r e s s e d a n d t h e r e s u l t a n t r e c o m b i n a n t p r o t e i n s u s e d in b i o c h e m i c a l a n d i m m u n o l o g i cal studies. A n t i b o d i e s c o u l d a l s o b e raised against t h e s e p r o t e i n s for u s e in l o c a l i z a t i o n a n d t i m e - c o u r s e e x p e r i m e n t s , a n d hopefully to s c r e e n the c D N A libraries o f o t h e r parasitic n e m a t o d e s .
O n c e parasitic n e m a t o d e h o m o l o g u e s o f cut-l a n d cut-2 h a v e b e e n fully c h a r a c t e r i z e d , t h e r o l e s a n d f u n c t i o n s o f t h e s e clearly important p r o t e i n s will b e b e t t e r u n d e r s t o o d a n d hopefully the k n o w l e d g e c a n in s o m e w a y b e u s e d in the c o n t r o l o f the d i s e a s e s c a u s e d by t h e s e parasitic n e m a t o d e s .
REFERENCES
LASSANDRO F . , SKBASTIANO M . , ZEI F . and BAZZICAUIPO P. : cut-2, a second cuticlin gene of Caenorbabditis elegans. The role of dity
rosine formation in the crosslinking of its product. Mot. Biocbem.
Parasitol, 1993, submitted.
POLITZ S . M . and PHILIPI- M . : Caenorbabditis elegans as a model for parasitic nematodes : A focus on the cuticle. Parasitol. Today, 1992, 8, 6-12.
SEBASTIA.NO M . , LASSANDRO F . , B A Z Z I C A U I P O P . : cut-l, a Caenorbabditis elegans gene coding for a dauer-specific non col
lagenous component of the cuticle. Dev. Biol., 1991, 146, 519- 530.
N E U R O N S A N D G E N E S I N V O L V E D I N CHE
MICAL S E N S I T I V I T Y I N N E M A T O D E S
BAZZICALUPO P.*, HILLIARD M., LEWIS E.*, D E R I S O L., S E B A S T I A N O M.*, R I S T O R A T O R E F.*
KEYWORDS : chemoreception. nematodes, behavior, avoidance, amphid.
SUMMARY
Organelles and neurons of nematodes involved in sensing chemical signals present in the environment are described. Laser ablation of neurons has helped assign them a specific function. Genetic mutatio
nal analysis has led to the identification of genes controlling the beha
viour of the worms and/or some cellular properties of the
chemosensory neurons. Some conclusions on the general organization and functioning of chemoreception in nematodes can be drawn.
h e m i c a l s i g n a l s from t h e e n v i r o n m e n t a r e t h e m o s t important s e n s o r y inputs for n e m a t o d e s . T h e ability to r e c e i v e a n d interpret c h e m i c a l s i g n a l s from t h e e n v i r o n m e n t is essential for parasitic n e m a t o d e s to find the host, c o m p l e t e t h e i r life c y c l e a n d r e p r o d u c e . I f b e t t e r u n d e r -
* International Institute of Genetics and Biophysics, CNR, via G.
Marconi 10, 80125 Napoli, Italy.
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/199401s1058
PARASITE B I O L O G Y AND BIOCHEMISTRY
s t o o d at the cellular a n d m o l e c u l a r level, c h e m o r e c e p t i o n w o u l d h a v e e n o r m o u s potential as a target for c h e m o t h e rapy a n d p o s s i b l y i m m u n o p r o t e c t i o n .
U n f o r t u n a t e l y , little is k n o w n a b o u t c h e m o r e c e p t i o n in parasitic n e m a t o d e s a n d its study in t h e s e o r g a n i s m s pre
s e n t s m a n y difficulties. H o w e v e r s o m e information is avai
l a b l e a b o u t c h e m o r e c e p t i o n in t h e free living n e m a t o d e C.
elegans, a n d m o r e i m p o r t a n t l y a variety o f e x p e r i m e n t a l a p p r o a c h e s are a p p l i c a b l e in t h e study o f this m o d e l orga
nism. A l t h o u g h n e m a t o d e s differ from e a c h o t h e r in size, life c y c l e , a n d o t h e r i m p o r t a n t a s p e c t s , t h e g e n e r a l p l a n a c c o r d i n g to w h i c h they are c o n s t r u c t e d is very c o n s e r v e d . T h u s a realistic strategy t o study c h e m o r e c e p t i o n in parasi
tic n e m a t o d e s is to start with C. elegans, with t h e aim o f e v e n t u a l l y using the k n o w l e d g e a n d t h e m o l e c u l a r r e a g e n t s a n d p r o b e s derived from this study, to identify the h o m o l o g o u s c o m p o n e n t s from parasitic s p e c i e s .
T h e m a i n c h e m o r e c e p t o r o r g a n s o f n e m a t o d e s a r e t h e A m p h i d s , o n e o n e a c h s i d e o f t h e h e a d . A c h e m o r e c e p t i o n f u n c t i o n is l i k e l y a l s o for t h e t w o P h a s m i d s a n d t h e s i x I n n e r L a b i a l N e u r o n s . As in o t h e r n e m a t o d e s , s e n s o r y o r g a n s in C. elegans are e a c h m a d e o f t w o n o n n e u r o n a l s u p p o r t cells, t h e s h e a t h cell a n d t h e s o c k e t cell that form a c h a n n e l a r o u n d a b u n d l e o f ciliated n e u r o n a l e n d i n g s ; in t h e c a s e o f c h e m o s e n s o r y n e u r o n s , t h e e n d i n g s r e a c h t h e e x t e r i o r t h r o u g h a g a p in t h e cuticle. In C. elegans the c e l lular a n a t o m y o f t h e s e o r g a n s a n d t h e p r e c i s e a n c e s t r y o f t h e cells contributing to t h e m h a v e b e e n d e s c r i b e d . Caenorhabclitis elegans b e h a v i o u r s i n f l u e n c e d by c h e m i c a l stimuli p r e s e n t in t h e e n v i r o n m e n t i n c l u d e : orientation in gradients o f a n u m b e r o f c h e m i c a l s acting as attractants o r r e p e l l e n t s ; a v o i d a n c e o f s o m e c h e m i c a l s acting as a r e p e l lent ; entry a n d exit from t h e d a u e r larvae d e v e l o p m e n t a l p a t h w a y ; e g g laying ; attraction o f m a l e s but not h e r m a p h r o d i t e s t o h e r m a p h r o d i t e s . W e h a v e c o n c e n t r a t e d o u r studies o n t h e a v o i d a n c e r e s p o n s e . W h e n it e n c o u n t e r s a r e p e l l e n t c h e m i c a l . C. elegans s t o p s a n d inverts its m o v e m e n t , b a c k i n g up. T h i s r e s p o n s e is relatively s i m p l e and, at least in principle, d o e s n o t require t h e integration o f infor
m a t i o n f r o m d i f f e r e n t s i g n a l s o r f r o m t h e s a m e s i g n a l v a r y i n g in t i m e o r s p a c e . As for o t h e r c h e m o r e c e p t i o n d e p e n d e n t b e h a v i o u r , laser ablation e x p e r i m e n t s have led t o t h e identification o f t h e m a i n s e n s o r y n e u r o n s involved in this r e s p o n s e ; they are t w o n e u r o n pairs, ASH a n d ADL, w h i c h s e n d their ciliated s e n s o r y termination t o t h e a m p h i d c h a n n e l . ASH a n d ADL are a l s o t h e o n l y t w o a m p h i d n e u r o n s s y n a p s i n g directly o n t o AVA, t h e m a i n i n t e r n e u r o n dri
ving b a c k w a r d m o v e m e n t . It is p o s s i b l e therefore that t h e a v o i d a n c e r e s p o n s e is m e d i a t e d b y a relatively s i m p l e three e l e m e n t circuit.
G e n e t i c m u t a t i o n a l a n a l y s i s is, in C. elegans, e x t r e m e l y powerful a n d is t h e staring point from w h i c h t o arrive at t h e m o l e c u l a r c l o n i n g o f g e n e s ; it h a s t h e r e f o r e already- b e e n u s e d in t h e study o f c h e m o r e c e p t i o n . T h e m u t a n t s that h a v e b e e n i s o l a t e d s o far c a n b e divided into t w o main g r o u p s :
i) m u t a n t s isolated o n t h e b a s i s o f alterations in b e h a v i o u r that d e p e n d o n t h e r e c e p t i o n o f c h e m i c a l s i g n a l s . T h e s e i n c l u d e m u t a n t s a b n o r m a l for c h e m o t a x i s , che, osmotic- a v o i d a n c e , osm, d a u e r formation, daf a n d volatile c h e m i c a l sensitively odr.
ii) D y f mutants. C h e m o s e n s o r y n e u r o n s o f live wild type C.
elegans t a k e u p F l u o r e s c e i n e I s o t h i o c y a n a t e ( F I T C ) from t h e m e d i u m . F o r wild type w o r m s , c o n d i t i o n s h a v e b e e n found u n d e r w h i c h o n l y s i x n e u r o n s o f e a c h a m p h i d a n d t h e t w o n e u r o n s o f e a c h p h a s m i d t a k e up t h e dye, trans
port it retrogradely a n d b e c o m e fluorescent. Mutants with alterations in this staining pattern h a v e b e e n isolated {dyf, for d y e filling a l t e r a t i o n ) a n d h a v e b e e n s h o w n t o h a v e various c h e m o s e n s o r y defects. T h e main a d v a n t a g e s o f this p h e n o t y p e are that it c a n b e d e t e c t e d o n single live w o r m s , m a k i n g g e n e t i c a n a l y s i s o f m u t a n t s faster ; a n d that t h e p h e n o t y p e is largely d e p e n d e n t o n s p e c i f i c p r o p e r t i e s o f c h e m o s e n s o r y n e u r o n s a n d thus c a n g e n e r a t e c h e m o r e c e p tion specific mutants. Mutations in m a n y o f t h e b e h a v i o u r a l m u t a n t s a l s o alter FITC u p t a k e , resulting in u n s t a i n e d o r p o o r l y stained animals.
W e h a v e isolated 19 D y f mutants a n d h a v e studied s o m e b a s i c a s p e c t s o f their b e h a v i o u r . T h e sensitivity most often lost in t h e s e mutants is t o w a r d s the p r e s e n c e o f high o s m o tic strength in t h e m e d i u m . All t h e mutants s h o w n o r m a l r e s p o n s e s t o t o u c h , i n d i c a t i n g that t h e y a r e m u t a t e d in g e n e s specifically r e q u i r e d for c h e m o r e c e p t i o n a n d are n o t g e n e r a l mutants o f t h e n e r v o u s system.
W e h a v e a l s o studied in m o r e detail dyf-1 a n e w g e n e o n LGI. B e s i d e s t h e l a c k o f s t a i n i n g o f c h e m o s e n s o r y c e l l s , mn335, t h e dyf-1 a l l e l e w e h a v e s t u d i e d , r e s p o n d s v e r y p o o r l y to high o s m o t i c strength, S D S a n d C u+ +. H o w e v e r its ability to avoid garlic a n d a r e p e l l e n t p r e s e n t in w o r m e x t r a c t s s e e m s u n a l t e r e d , as is its c a p a c i t y to r e s p o n d t o light t o u c h . Ultrastructurally t h e m a i n defect o f mn335 is shorter s e n s o r y cilia a n d a c o l l a p s e d amphidial c h a n n e l . W e h a v e m a p p e d t h e g e n e t o L i n k a g e G r o u p /, n e a r unc-38 a n d h a v e started a project to c l o n e it molecularly.
D y f mutants, i n c l u d i n g dyf-1 , identify g e n e s i n v o l v e d in t h e g e n e r a l p h y s i o l o g y o f t h e c h e m o r e c e p t o r n e u r o n s a n d / o r o f the s u p p o r t c e l l s ; their p r o d u c t s are n e c e s s a r y for t h e u p t a k e o f t h e d y e from t h e m e d i u m a n d the retro
g r a d e transport t o t h e cell b o d y . G e n e s c o d i n g for t h e s p e cific r e c e p t o r m o l e c u l e s l o c a t e d o n t h e n e u r o n a l e n d i n g s , a n d for t h e c o m p o n e n t s o f t h e c h e m o - s e n s o r y transduction p a t h w a y r e m a i n largely u n d e t e c t e d in this t y p e o f s c r e e n . In o r d e r to identify t h e s e t y p e s o f g e n e w e h a v e r e c e n t l y started to isolate b e h a v i o u r a l mutants with t h e aim o f fin
ding t h o s e specifically u n a b l e t o s e n s e a n d a v o i d certain c h e m i c a l s but still c a p a b l e o f s e n s i n g others. W e h a v e isola
t e d several mutants a n d are characterizing t h e m g e n e t i c a l l y a n d b e h a v i o r a l l y . W e h a v e u s e d stringent b e h a v i o r a l assays in the s c r e e n i n g stage a n d it is interesting that m o s t o f the mutants isolated d o not s h o w a D y f p h e n o t y p e . T h i s result indicates that it is p o s s i b l e to isolate mutants in a different set o f g e n e s w h i c h c o u l d i n c l u d e t h e r e c e p t o r s a n d t h e c h e m o s e n s o r y p a t h w a y .
T h e a n a t o m i c a l studies, t h e laser ablation e x p e r i m e n t s a n d t h e b e h a v i o u r a l a n d ultrastructural analysis o f t h e mutants i s o l a t e d a r e p r o v i d i n g i m p o r t a n t i n f o r m a t i o n s r e g a r d i n g s o m e b a s i c features o f c h e m o r e c e p t i o n in n e m a t o d e s : 1. E a c h c h e m o s e n s o r y n e u r o n b e a r s r e c e p t o r s for m o r e t h a n o n e c h e m i c a l .
2. R e c e p t o r s for c h e m o a t t r a c t a n t a n d for d a u e r formation are p r e s e n t o n m o r e than o n e c h e m o s e n s o r y n e u r o n . 3. Most b e h a v i o r a l mutants, a l t h o u g h isolated o n t h e basis
• Parasite. 1994. / . I S 59
PARASITE BIOLOGY AND BIOCHEMISTRY
o f a single altered b e h a v i o u r , a r e p l e i o t r o p i c a n d therefore d e f e c t i v e in o t h e r c h e m i c a l sensitivities.
4. T h e D y f p h e n o t y p e is a s s o c i a t e d with t h e l o s s o f sensiti
vity t o m a n y different c h e m i c a l s . Alterations in t h e s h a p e o r matrix c o n t e n t o f t h e amphidial c h a n n e l a l s o result in a D y f p h e n o t y p e .
5. W h i l e w a t e r s o l u b l e c h e m i c a l s s e e m t o h a v e r e c e p t o r s o n the n e u r o n s w h o s e cilia a r e e x p o s e d t o t h e o u t s i d e (ASH a n d A D D , attraction t o a n d repulsion b y volatile c h e m i c a l s are m e d i a t e d b y r e c e p t o r s p r e s e n t o n t h e amphidial wing cells AWA, AWES a n d A W C .
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CHALFIE M. and WHITE J.-G. : The nervous system in The Nematode Caenorbabditis elegans. Wood W . B . (ed.), Cold Spring Harbor Lab. C . S . H . , New York, 1988, 337-391.
WHITE J . G . , SOUTHGATE E., THOMSON J . N . and BRENNER S. : The struc
ture of the nervous system o f the nematode Caenorbabditis ele
gans. Philos. Trans. R. Soc. London, Ser. B., 1986, 314, 1-340.
N - A C E T Y L A T I O N O F P O L Y A M I N E S A N D BIOGENIC A M I N E S I N PARASITIC N E M A T O D E S
AISIEN S.O.*, DAVIDS G**, HELLMUND C**, NIEMANN G.**
AND WALTER R.D.**
KEYWORDS Polyamines. biogenic amines. N-acetylase. nematodes.
T
h e p r e v i o u s l y d e s c r i b e d p o l y a m i n e N - a c e t y l a s e from Fasciola bepatica h a s b e e n o b s e r v e d to have an additional function, t h e acetylation o f b i o g e n i c a m i n e s (Aisien a n d W a l t e r , 1 9 9 2 , 1 9 9 3 ) . It w a s c o n c l u d e d that N - a c e t y l a t i o n plays a major role in t h e a m i n e m e t a b o l i s m o f trematodes. In continuation o f t h e s e o n g o i n g studies o n t h e p r o c e s s o f N- acetylation in parasitic helminths, w e h a v e d e t e c t e d b i o g e n i c a m i n e acetylation in t h e tissue dwelling filaria Onchocerca volvulus a n d t h e intestinal parasite Ascaris suum. T h e aim o f o u r study w a s t o a s c e r t a i n if o u r p r e v i o u s finding that a single e n z y m e is r e s p o n s i b l e for d i a m i n e , p o l y a m i n e a n d b i o g e n i c a m i n e acetylation in trematodes, is a feature c o m m o n to all h e l m i n t h s . Results from investigation using A.
suum indicate that t w o i n d e p e n d e n t e n z y m e s are respecti
vely r e s p o n s i b l e for p o l y a m i n e a n d b i o g e n i c a m i n e acetyla
t i o n . C h r o m a t o g r a p h y o f t h e 1 0 0 , 0 0 0 g s u p e r n a n t o n DEAE-cellulose r e v e a l e d t w o e n z y m e activity p e a k s , b o t h o f w h i c h have activity for histamine. T h e first p e a k w a s o b s e r v e d to c a t a l y s e o n l y b i o g e n i c a m i n e acetylation w h i l e t h e s e c o n d a c e t y l a t e d p u t r e s c i n e a n d o t h e r d i a m i n e s . O n t h e basis o f the Km- v a l u e s o b t a i n e d with b o t h e n z y m e s for hista
m i n e , p e a k II s e e m s t o b e m o r e specific for this substrate
* Department of Zoology, University of Benin, Benin-City, Nigeria.
** D e p a r t m e n t o f B i o c h e m i c a l Parasitology, Bernhard-Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany.
with a Km- v a l u e o f 3 7 uM c o m p a r e d to p e a k I w h i c h h a d a Km- v a l u e o f 500LIM. In contrast t h e Km- v a l u e s o b t a i n e d for b i o g e n i c a m i n e s with p e a k I w e r e as follows : T y r a m i n e , 0 . 9 uM, tryptamine, 1.7 uM, o c t o p a m i n e , 2 9 uM, serotonin, 9 . 5 uM a n d ß-phenylethylamine, 1.5 uM. Epinephrine, n o r e p i n e phrine a n d d o p a m i n e w e r e n o t physiological substrates for t h e N - a c e t y l a s e . T h e e n z y m e h a s a m o l e c u l a r m a s s o f a p p r o x i m a t e l y 3 0 k D a a n d w a s slightly inhibited b y c o e n z y m e A, a product o f t h e acetylation p r o c e s s . T h e specificity o f p e a k II for diamines a n d histamine a n d its lack o f activity for p o l y a m i n e s indicates that this e n z y m e is most probably the novel putrescine acetylase previously reported b y Wittich and Walter ( 1 9 9 0 , 1 9 9 1 ) from O. volvulus a n d A. suum.
A S S E S S M E N T O F THE POLYAMINE M E T A B O L I S M O F FILARIAL W O R M S A S A T A R GET F O R C H E M O T H E R A P Y
MÜLLER S.*, H U N T E R K.J.**, K O N D U R S*, HELLMUND C.*, FAIRLAMB A.H.** & WALTER R.D.*
KEYWORDS : Polyamines. metabolism, chemotherapy, filaria. N-acetyltransfe- rase. polyamine oxidase. S-adenosylmethionine decarboxylase.
Polyamines a r e essential for t h e proliferation a n d diffe
rentiation o f c e l l s a n d o r g a n i s m s . T h e p r e s e n c e o f poly
a m i n e s h a s b e e n d e m o n s t r a t e d in Onchocerca volvulus a n d a l l i e d p a r a s i t e s a n d t h e i n v e s t i g a t i o n o f t h e p o l y a m i n e m e t a b o l i s m h a s i d e n t i f i e d u n u s u a l p a t h w a y s w h i c h a r e p o t e n t i a l c h e m o t h e r a p e u t i c t a r g e t s . T h e s e p a t h w a y s a r e crucial for parasite survival a n d differ in s o m e b i o c h e m i c a l a s p e c t s from t h e h o s t c o u n t e r p a r t s , thus a l l o w i n g for t h e rational d e s i g n o f drugs for a parasite-specific c h e m o t h e r a p e u t i c attack. A p r o p o s e d s c h e m e o f t h e p o l y a m i n e m e t a b o l i s m in filarial w o r m s is s h o w n in Figure 1.
S i n c e t h e p r e s e n c e o f o r n i t h i n e d e c a r b o x y l a s e ( O D C ) acti
vity is q u e s t i o n a b l e , t h e initial s t e p in t h e b i o s y n t h e s i s o f p o l y a m i n e s a p p e a r s t o b e l a c k i n g a n d t h u s t h e p a r a s i t e d e p e n d s o n u p t a k e from t h e host for its p o l y a m i n e supply.
An i n t e r c o n v e r s i o n p a t h w a y f o r p o l y a m i n e s h a s b e e n d e m o n s t r a t e d w h i c h d o e s n o t i n v o l v e t h e p o l y a m i n e N - a c e - tyltransferase, t h e rate limiting s t e p in t h e i n t e r c o n v e r s i o n o f p o l y a m i n e s in m a m m a l s . A n o v e l t y p e o f p o l y a m i n e o x i d a s e h a s b e e n identified w h i c h is s o l e l y r e s p o n s i b l e for t h e i n t e r c o n v e r s i o n a n d d e g r a d a t i o n o f p o l y a m i n e s . In addition, a p a r a s i t e - s p e c i f i c N - a c e t y l t r a n s f e r a s e for p u t r e s c i n e w a s found w h i c h is involved in t h e d e g r a d a t i o n a n d e x c r e t i o n o f e x c e s s p o l y a m i n e s .
D e p l e t i o n o f p o l y a m i n e levels in filarial w o r m s is thought t o h a v e c y t o s t a t i c i f n o t c y t o t o x i c e f f e c t s , a s h a s b e e n s h o w n previously in t h e c h e m o t h e r a p y o f c a n c e r a n d p r o t o z o a n infections b y D L - a - d i f l u o r o m e t h y l o r n i t h i n e ( D F M O ) . Filarial w o r m s a n d o t h e r h e l m i n t h s l a c k o r n i t h i n e d e c a r b o x y l a s e , t h e t a r g e t f o r D F M O . T h u s , a s t h e p a r a s i t e d e p e n d s o n t h e u p t a k e o f p o l y a m i n e s from t h e h o s t tissues, inhibition o f transport m e c h a n i s m s resulting in rapid d e p l e tion o f p o l y a m i n e s s h o u l d b e e x p l o i t a b l e for c h e m o t h e r a p y .
* D e p a r t m e n t o f B i o c h e m i c a l Parasitology, B e r n h a r d Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg. Germany.
** Department o f Medical Parasitology. London School o f Hygiene
& Tropical Medicine, Keppel St, London, WC1E 7HT, U.K.
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