Molecular transformation of myoblasts induced by excretory/secretory products of trichinella...
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Poster Sessions / Parasitology International 47 (Syppl.) (1998) 283-389 339 P-081@ STA GESJ’JXJWC JSMYMES OFASCARJS SZJ.?J.M COh4PLEX II: THE FUMARATE REDUCTASE OF THE PARASITIC ADULT AND THE SUCCINATE DEHYDROOENASE OF FREE-LIVING LARVAE HAVETHECOMMONIRON-SULFURSUBUNIT m ft*. Hirawake H.*, Saruta F.*, Takatniya S.**, Kojima S.*** and Kita K.* *Depar&ment of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, **Department of Parasitology, Juntendo University, School of Medicine, Tokyo and ***Dfzlpaiunem OS ?ar&tdrogy,~ne in&irute dr me&d Xlence, The Unirve~&~~~d~~.~d~o.~~~ Mitochondrial respiratory chain of the parasitic nematode, &caris surtm, changes dramatically during the life cycle. Complex II from mitachondria of the adult A. suum exhibits high ftunarakz tiuctase (FRD) activity and plays a key role in the anaerobic metabolism. In contrasi, complex II from free-living sewnd stage larva (L2) shows much lower FRD activity than that from adult, and functions as succiniare ~&Y~EQJSWS ~?53fQ. *We ~~~ou+$Y r-qpeb timx3 evidence br ih& r&&FSce Of the tW0 di~trnCS ~tage-~peofiC isozymes of complex II in A. SttYm by the biochemical analysis of the isolated camplc-& /+ml. %Qw. ?tId 72 w&iw!Pa&& 2nd Yu$fg&& ‘hat. w. least the Fp and the cybS subunit of larval complex II were different from those of adult complex II (Saruta er al. (1995) J. Eiol. Chem. 270, !Z8] In the present study, larval complex II was partially purified on DEAE-celIuIotine cdumn chromatography. And theIp subunit purified from peak fractions by gel electrophoresis was electroblottcd onto PVDFmembmne, and then sequenced directly. The N-terminal twenty six amino acid sequence was identical to the mature protein of theadult Ipsubunit. Thepeptidemapbylimitedproteolysis of the Ip extmcled from larval mitochondria was almost identical with the adult Ip. In a&&w, e $b&iz&a~skwi& expxw&d .i&z& >p subunit in the free-living second stage larvae. These results suggest that stage-specific isuzymes of A. SUMI complex II have the common iron-sulfur subunit, even though complex II of adult functions as a fumarak r&me anb tia r5 5arvae a suc&nale dehydrogenase. p-0811 COMPARISON OF BIOCHEMICAL PROPERTIES OF ASCARLSSVLBf ACID PRO’I’EINASE AND DIROlVLARL4 -ISASPARTIC PROTEINASE Sate K*, Kano S**, Oue M ** and Suzuki M” *Department of Laboratory Sciences, Gunma University School of Health Sciences, Maebashi, Japan and ** Department of Parasitology, Gunma University School of Medicine, Maebashi, Japan Parasite proteinases are thought to play a* important role for the parasite’s nutritional metabolism and physiological function. Many woigs ‘nave ‘men pdtii&~h reg.&img ‘tnese e~vrt~es “room parasitic belmintbes and protozoa. We have reported the biochemical properties of purilied D. tinmitis aspartic proteinase. The immunohistochemical studies revealed the localization of the enzyme in the worm tissue. In the present study, we describe the details of partial purification of Aacaris suum acid proteinase and it’s biochemical characteristics, and then comparison of A. euutn acid pmteinaae and A immitis aepartic proteinase. A. euum acid pmteinase was partially purified 83-fold by a chromatography on DEAE-sephadex followed by pepstatin-agarose gel affinity chromatography. Optimum pH of the enzyme was in the range of pH 3.2 -3.8 and Km value obtained from Lineweaver Burk plot was about %23 &X? ?XV lm&V%&i- we-+-L? & l&7-c?rymV %a%- &o%t+ 66,000 by SDS-PAGE. The proteinaee showed a specific activity against human hemoglobin and bovine hemoglobin although it showed a weak activity against equine myoglobin. Pepstatin, a potent inhibitor of aepartic proteinases, inhibited about 90% of the activity by the concentration of 5 x 1Oa M. Optimum pH of D. imm.itie aspartic pmteinase was 2.8-3.4 and the molecular weight of the enayme iv88 about 42,lXQ D. i-&4 proteinase &owed a potent activity against human hemoglobin and equine myoglobin. Comple* &&.itian d t& pm*&-t& B&v<~ hy pepeta& wan obearvad in the order of lo” M. Small differences were observed in the biochemical properties of both enzymes, however, anti-D. immifiaproteinase antibody showed a cross-reaction to A. suum acid pmt8inaee. P-I%12 THE SEARCH OF NEW MOLECULAR MARKERS FOR THE PARASITIC WORMS OF TRICHINELLA SPECIES IDENTIFY Chrisanphova G.G.*, Manoylenko D.A.*, Zhilin A.V.*, Ryskov A.P.*, Asatrian A.M.**, Movsessian S.O.**, Semyenova S.K.* The random amplified polymorphic DNA (RAPD) technique was used for genome analysis of two Trichinella species (T. spiralis and T. qseudospiralisl ,. wl~_kh QbtaiQp2 0222 *_<f*c*qtr rY0.95 animals tinice ( rat, ratzit, chicken\. RAPI3 products were cloned in the pMOS Blue-T vector and 50 recombinant clones were PID- duced. The size of inserts ranged from 0,s to 1,5 kb. DNA of 10 recombinant clones were iso- lated, particularly sequenced and then used as a probes in blot-hybridization experiments with genome DNA and WD-patterns of Tri- chinella species (isolates). The species spe- cific and host specific clones were revealed. High nucleotide sequence homology between Tri- chinella sp. and C. elegans was found. The particular similarities were found across Tri- chinella qenome and human.diseases vi-ruses. P-0813 MOLECULAR TRANSFORMATION OF MYOBLASTS INDUCED BY EXCRETORY/SECRETORY PRODUCTS OF TRICHINELLA SPIRALIS Mak. C.H. and Ko, R.C.C. Department of Zoology, The University 6f Hong Kong , Hong QJ%Qina bichineh spin& i3 an inuacellular parasitethat invades terminally differentiated mamroalian muscles,modifying them into a new synqtium know as the nurse cell. la&ted skeletal muscles are biggemd to dediff~tiatc and n@c spxific gems expressiw are down reg&bzd. Reumly , we have ohm that me cxcretorylxcretoly (Ee) producb of the infective-stage larvae cao initiate the reorganization of myocytes.In vifro studiesrevealedthattbe US products may modify the p&tern of host gene cxpmssion. This was indicated by tbc induction of type IV collageo expression in treated myoblasts. Using a 2Gmer random oligonucleotide as the target DNA in electromobility shift assay (EMSA), we were able to show that doubleslmnded Lose activity was present in the US pmducts. In &dux, ti y&- no.%%+* w :w zx% _q$&?&& .illRt _x+&& biidii protein(s) may be present in both T. pseudospira~is and Z spirdis E’S products The DNA bindingfactor(s) in the US products of both species botmd to the targetDNAs, eitherwith similar specificity.or with low specificity. US products 6’omT.spiralis boundto the targetDNAs of bomologws and beterologous specieswithsimilar affinity,and vice versa Nuck;lr factor@) may serve as tbe effector molecules that modify the genomic expression of host muscles.
Transcript of Molecular transformation of myoblasts induced by excretory/secretory products of trichinella...
Poster Sessions / Parasitology International 47 (Syppl.) (1998) 283-389 339
P-081@ STA GESJ’JXJWC JSMYMES OFASCARJS SZJ.?J.M COh4PLEX II: THE FUMARATE REDUCTASE OF THE PARASITIC ADULT AND THE SUCCINATE DEHYDROOENASE OF FREE-LIVING LARVAE HAVETHECOMMONIRON-SULFURSUBUNIT
m ft*. Hirawake H.*, Saruta F.*, Takatniya S.**, Kojima S.*** and Kita K.* *Depar&ment of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, **Department of Parasitology, Juntendo University, School of Medicine, Tokyo and ***Dfzlpaiunem OS ?ar&tdrogy,~ne in&irute dr me&d Xlence, The Unirve~&~~~d~~.~d~o.~~~
Mitochondrial respiratory chain of the parasitic nematode, &caris surtm, changes dramatically during the life cycle. Complex II from mitachondria of the adult A. suum exhibits high ftunarakz tiuctase (FRD) activity and plays a key role in the anaerobic metabolism. In contrasi, complex II from free-living sewnd stage larva (L2) shows much lower FRD activity than that from adult, and functions as succiniare ~&Y~EQJSWS ~?53fQ. *We ~~~ou+$Y r-qpeb timx3
evidence br ih& r&&FSce Of the tW0 di~trnCS ~tage-~peofiC isozymes of complex II in A. SttYm by the biochemical analysis of the isolated camplc-& /+ml. %Qw. ?tId 72 w&iw!Pa&& 2nd Yu$fg&& ‘hat. w. least the Fp and the cybS subunit of larval complex II were different from those of adult complex II (Saruta er al. (1995) J. Eiol. Chem. 270, !Z8]
In the present study, larval complex II was partially purified on DEAE-celIuIotine cdumn chromatography. And the Ip subunit purified from peak fractions by gel electrophoresis was electroblottcd onto PVDFmembmne, and then sequenced directly. The N-terminal twenty six amino acid sequence was identical to the mature protein of theadult Ipsubunit. Thepeptidemapbylimitedproteolysis of the Ip extmcled from larval mitochondria was almost identical with the adult Ip. In a&&w, e $b&iz&a~skwi& expxw&d .i&z& >p subunit in the free-living second stage larvae. These results suggest that stage-specific isuzymes of A. SUMI complex II have the common iron-sulfur subunit, even though complex II of adult functions as a fumarak r&me anb tia r5 5arvae a suc&nale dehydrogenase.
p-0811 COMPARISON OF BIOCHEMICAL PROPERTIES OF ASCARLSSVLBf ACID PRO’I’EINASE AND DIROlVLARL4 -ISASPARTIC PROTEINASE
Sate K*, Kano S**, Oue M ** and Suzuki M” *Department of Laboratory Sciences, Gunma University School of Health Sciences, Maebashi, Japan and ** Department of Parasitology, Gunma University School of Medicine, Maebashi, Japan
Parasite proteinases are thought to play a* important role for the parasite’s nutritional metabolism and physiological function. Many woigs ‘nave ‘men pdtii&~h reg.&img ‘tnese e~vrt~es “room parasitic belmintbes and protozoa. We have reported the biochemical properties of purilied D. tinmitis aspartic proteinase. The immunohistochemical studies revealed the localization of the enzyme in the worm tissue. In the present study, we describe the details of partial purification of Aacaris suum acid proteinase and it’s biochemical characteristics, and then comparison of A. euutn acid pmteinaae and A immitis aepartic proteinase. A. euum acid pmteinase was partially purified 83-fold by a chromatography on DEAE-sephadex followed by pepstatin-agarose gel affinity chromatography. Optimum pH of the enzyme was in the range of pH 3.2 -3.8 and Km value obtained from Lineweaver Burk plot was about %23 &X? ?XV lm&V%&i- we-+-L? & l&7- c?rymV %a%- &o%t+ 66,000 by SDS-PAGE. The proteinaee showed a specific activity against human hemoglobin and bovine hemoglobin although it showed a weak activity against equine myoglobin. Pepstatin, a potent inhibitor of aepartic proteinases, inhibited about 90% of the activity by the concentration of 5 x 1Oa M. Optimum pH of D. imm.itie aspartic pmteinase was 2.8-3.4 and the molecular weight of the enayme iv88 about 42,lXQ D. i-&4 proteinase &owed a potent activity against human hemoglobin and equine myoglobin. Comple* &&.itian d t& pm*&-t& B&v<~ hy pepeta& wan obearvad in the order of lo” M. Small differences were observed in the biochemical properties of both enzymes, however, anti-D. immifiaproteinase antibody showed a cross-reaction to A. suum acid pmt8inaee.
P-I%12
THE SEARCH OF NEW MOLECULAR MARKERS FOR THE PARASITIC WORMS OF TRICHINELLA SPECIES IDENTIFY
Chrisanphova G.G.*, Manoylenko D.A.*, Zhilin A.V.*, Ryskov A.P.*, Asatrian A.M.**, Movsessian S.O.**, Semyenova S.K.*
The random amplified polymorphic DNA (RAPD) technique was used for genome analysis of two Trichinella species (T. spiralis and T. qseudospiralisl ,. wl~_kh QbtaiQp2 0222 *_<f*c*qtr rY0.95 animals tinice ( rat, ratzit, chicken\. RAPI3 products were cloned in the pMOS Blue-T vector and 50 recombinant clones were PID- duced. The size of inserts ranged from 0,s to 1,5 kb. DNA of 10 recombinant clones were iso- lated, particularly sequenced and then used as a probes in blot-hybridization experiments with genome DNA and WD-patterns of Tri- chinella species (isolates). The species spe- cific and host specific clones were revealed. High nucleotide sequence homology between Tri- chinella sp. and C. elegans was found. The particular similarities were found across Tri- chinella qenome and human. diseases vi-ruses.
P-0813
MOLECULAR TRANSFORMATION OF MYOBLASTS INDUCED BY EXCRETORY/SECRETORY PRODUCTS OF TRICHINELLA SPIRALIS
Mak. C.H. and Ko, R.C.C.
Department of Zoology, The University 6f Hong Kong , Hong QJ%Qina
bichineh spin& i3 an inuacellular parasite that invades terminally differentiated mamroalian muscles, modifying them into a new synqtium know as the nurse cell. la&ted skeletal muscles are biggemd to dediff~tiatc and n@c spxific gems expressiw are down reg&bzd. Reumly , we have ohm that me cxcretorylxcretoly (Ee) producb of the infective-stage larvae cao initiate the reorganization of myocytes. In vifro studies revealed that tbe US products may modify the p&tern of host gene cxpmssion. This was indicated by tbc induction of type IV collageo expression in treated myoblasts. Using a 2Gmer random oligonucleotide as the target DNA in electromobility shift assay (EMSA), we were able to show that double slmnded Lose activity was present in the US pmducts. In &dux, ti y&- no.%%+* w :w zx% _q$&?&& .illRt _x+&& biidii protein(s) may be present in both T. pseudospira~is and Z spirdis
E’S products The DNA binding factor(s) in the US products of both species botmd to the target DNAs, either with similar specificity. or with low specificity. US products 6’om T.spiralis bound to the target DNAs of bomologws and beterologous species with similar affinity, and vice versa Nuck;lr factor@) may serve as tbe effector molecules that modify the genomic expression of host muscles.
