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34 Lahiri and Bhattacharya9+2 microtubular arrangement. The basal body as well To isolate and purify the PFR proteins, the firstas the base of the flagellum remains ensheathed by a essential step was to successfully separate the flagellamembrane, and the central rod of the flagellum from intact cell bodies. The process of flagellaemerges freely. Centrifugation at 2600 x g detached isolation was monitored by using SEM to make surethe flagellum partially from the basal body. Complete that most of the cells remained intact and only lostdetachment was obtained at 3200 x g but the flagellum their flagella. Because the promastigotes werewas still surrounded by a membrane. Non-ionic obtained by centrifuging the culture medium at 2000 xdetergent treatment removed the flagellar g, the cells were subjected to three different highermembranes.centrifugation speeds starting from 2600 x g andDISCUSSIONincreasing gradually to 3700 x g to isolate the flagella.When observed under a SEM, it was observed thatAmong protozoans, the kinetoplastid trypanosomes most of the cells centrifuged at 3700 x g were rupturedhave always held an important position in the (Fig. 3), while those centrifuged at 2600 x g retainedscientific research scenario, not only for their medical their flagella (Fig. 1). Thus, the optimumimportance but also for possessing diverse cellular centrifugation speed for successful isolation ofprocesses. One such unique cytoskeletal structure is flagella was chosen to be 3200 x g (Fig. 2).PFR. The canonical 9+2 axoneme of the flagellumThe flagellar membranes were removed by treatmentinitiates beating of the latter in most eukaryotes, butwith the non-ionic detergent Nonidet P-40. It latterthe elaborate PFR structure has been observed in onlyappears to have no effect on the integrity of thekinetoplastids, euglenoids and some dinoflagellates.flagellar cytoskeleton and is, therefore, unable toTill date, the PFR and its proteins, namely PFR1 and break the attachment between the PFR and axonemePFR2 have been studied in T. cruzi, T. brucei, L. (Fig. 5). Beacuse it has been determined that the linksmexicana and L. amazonensis. These studies revealed between PFR and axoneme are highly sensitive tothat PFR is essential for proper parasite motility and trypsin (Moreira-Leite et al., 1999), the detachment ofviability, and both these functions are directly PFR from axoneme was accomplished by proteolyticattributable to the PFR proteins – PFR1 and PFR2 treatment.(Santrich et al., 1997; Bastin et al., 1998). Using newSDS-PAGE analysis showed two protein bands of molmolecular-genetic techniques, PFR1, PFR2 andwt 76 kDa and 68 kDa that are presumed to be twoPFR1/PFR2 null mutants of T. brucei and L. mexicanafractions of the PFR proteins (Fig. 4). Extensivehave been generated. The mutant cells lacked a nativestudies carried out till date on the major PFR proteinsPFR structure, showing that despite sharing over 60%in the parasitic haemoflagellates T. cruzi, T. brucei, L.amino acid homology and similar physical properties,mexicana and L. amazonensis have shown that thePFR1 and PFR2 are essential and significantlyPFR proteins migrate in SDS-PAGE as two bands withfunctional components of PFR. Mutants of L.mol wt of 70-75 kDa and 68-72 kDa, respectively, andmexicana having a PFR2-phenotype were unable toappear to be present in approximately equimolarcolonize in the vector's gut whereas PFR1 and/oramounts. So, this result is in conformity with thePFR2 mutants were viable in axenic culture. Thisresults reported in previous studies.indicates that the PFR plays a key role in the viabilityof Leishmania in a natural habitat (Hunger-Glaser and Transmission electron microscopy study revealed theSeebeck, 1997). Leishmania parasites lacking PFR ultrastructural details of PFR. The longitudinalwere found to display severe disturbances in flagellar section of a flagellum shows that the lattice-like PFRwaveforms e.g. reduced wavelength and amplitude runs parallel to the axoneme even before theand decrease in frequency as compared to the beating emergence of the flagellum from the flagellar pocketpatterns of normal parasites (Santrich et al., 1997). So, (Fig. 5). It also shows the attachment of the flagellumit is of prime importance to isolate and study the to the basal body at the anterior end of the parasite. Theultrastructure of the flagellum and PFR of L. axoneme of the flagellum showed typical eukaryoticdonovani, and to determine its role in the life cycle of 9+2 microtubular arrangement (Fig. 5). The basalthe parasite with special reference to PFR proteins. body as well as the base of the flagellum remainsThe present studies were carried out with an aim to ensheathed by a membrane and the central rod of thefulfill the above objectives.flagellum emerges freely. Centrifugation at 2600 x gdetached the flagellum partially from the basal body.
The PFR proteins of Leishmania donovani35Complete detachment was obtained at 3200 x g but theflagellum was still surrounded by a membrane. Nonionicdetergent treatment removed the flagellarmembranes.Bray RS. 1983. Leishmania: chemotactic responses ofpromastigotes and macrophages in vitro. J Protozool 30:322-329.Cachon J, Cachon M, Cosson M-P and Cosson J. 1988. Theparaflagellar rod: a structure in search of a function. BiolThe alternation of flagellate and nonflagellate forms Cell 63: 169-181.of the parasite is attributable to their needs forCunha NL, De Souza W and Hassón-Voloch A. 1984. Isolationinfection and survival within the host cells. The and characterization of the paraxial structure offlagellum is essential for attachment to macrophages, Herpetomonas megaseliae. J Submicrosc Cytol 16: 705-and subsequent penetration and stabilization within 713.macrophages. Apart from attachment, penetration andDe Souza W and Souto-Padron T. 1980. The paraxial structurestabilization within host cells, the main function of of the flagellum of trypanosomatidae. J Parasitol 66: 229-flagella is to confer motility and viability to cells so 236.that they can infect host cells. Once the infectionDeflorin J, Rudolf M and Seebeck T. 1994. The majorprocess is completed, the presence of flagella is nocomponents of the paraflagellar rod of Trypanosome bruceilonger required. So, the flagella become rudimentary are two similar, but distinct proteins which are encoded byand non-functional in the intracellular forms. two different gene loci. J Biol Chem 269: 28745-28751.The present study shows the successful isolation of Farina M, Attias M and Souto-Padron T. 1980. The paraxial rodflagella through scanning electron microscopy. It of trypanosomatids. J Protozool 33: 552-557.reports that PFR is composed of two major proteins Fouts DL, Stryker GA, Gorski KS, Miller MJ, Nguyen TV,(mol wt 76 kDa and 68 kDa) in L. donovani, which are Wrightsman RA and Manning JE. 1998. Evidence for fourunique to that structure and bear no significant distinct major protein components in the paraflagellar rod ofhomology to other known proteins. TransmissionTrypanosoma cruzi. J Biol Chem 273: 21846-21855.electron microscopy study of the flagellum and PFR Freymüller E and Camargo EP. 1981. Ultrastructuralprovides a relatively complete picture of the biology differences between species of trypanosomatids with andof these structures. It shows the position of the without endosymbionts. J Protozool 28: 175-182.axoneme and PFR within the flagellum, how they are Fuge H. 1969. Electron microscopic studies and theconnected to each other and their structural intraflagellar structure in trypanosomes. J Protozool 16:peculiarities. This study is an important guideline for 460-465.those who are carrying out researches on the flagellum Gallo JM and Schrevel J. 1985. Homologies betweenand PFR of L. donovani.paraflagellar rod proteins from trypanosomes andACKNOWLEDGEMENTSeuglenoids revealed by a monoclonal antibody. Eur J CellBiol 36: 163-168.The financial assistance from the Council of Scientificand Industrial Research, New Delhi, [scheme No. 60(0042)/01/EMR-II] is gratefully acknowledged.REFERENCESAraujo FG and Morein B. 1991. Immunization withTrypanosoma cruzi epimastigote antigens incorporated intoiscoms protects mice against lethal challenge in mice. InfectImmun 59: 2909-2914.Bastin P, Mattews KR and Gull K. 1996. The paraflagellar rodof kinetoplastida: solved and unsolved questions. ParasitolToday 12: 302-307.Bastin P, Sherwin T and Gull K. 1998. Paraflagellar rod is vitalfor trypanosome motility. Nature 391: 548.Beard CA, Saborio JL, Tewari D, Krieglstein KG, Henschen AHand Manning JE. 1992. Evidence for two distinct majorprotein components, PAR 1 and PAR 2, in the paraflagellarrod of Trypanosoma cruzi. J Biol Chem 267: 21656-21662.Hunger-Glaser I and Seebeck T. 1997. Deletion of the genes forthe paraglagellar rod protein PFR-A in Trypanosomabreecei is probably lethal. Mol Biochem Parasitol 90: 347-351.Hyams J. 1982. The Euglena paraflagellar rod: structure,relationship to other flagellar components and preliminarybiochemical characterization. J Cell Sci 55: 199-210.Imbodem M, Müller N, Hemphill A, Matioli R and Seebeck T.1995. Repetitive proteins from the flagellar cytoskeleton ofAfrican trypanosomes are diagnostically useful antigens.Parasitol 110: 249-258.Ismach R, Ciani CML, Caulfield JP, Langer PJ, Hein A andMcMahon-Pratt D. 1989. Flagellar membrane and paraxialrod proteins of Leishmania: characterization employingmonoclonal antibodies. J Protozool 36: 617-624.Killick-Kendrick R. 1979. Biology of Leishmania inphlebotomine sandflies. In: Biology of the Kinetoplastida.Lumsden WHR and Evans DA (Eds) Academic Press, NewYork. pp. 203-260.
Page 1 and 2: TISSN: 0971-7196Journal ofVolume 30
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Page 11 and 12: Haemonchus histochemistry, biochemi
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Page 18 and 19: 12 Soodconcentration (Kocher et al.
Page 20 and 21: 14 SoodKapur J and Sood ML. 1984b.
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Page 24 and 25: 18 Banyal and Elangbamin vitro. The
Page 26 and 27: 20 Banyal and Elangbamparasite in t
Page 28 and 29: 22 Banyal and Elangbamand presence
Page 30 and 31: 24 Banyal and ElangbamTable I: Mala
Page 32 and 33: 26 Banyal and Elangbamantibodies in
Page 34 and 35: 28 Banyal and ElangbamSchneider J,
Page 38 and 39: 32 Lahiri and Bhattacharyasurfaces
Page 42 and 43: 36 Lahiri and BhattacharyaKillick-K
Page 44 and 45: 38Edward and Bernardtopographical f
Page 46 and 47: 40Edward and Bernardfour villages b
Page 48 and 49: 42Sangwan and Sangwanvaccinated aga
Page 50 and 51: 44Sangwan and Sangwanpathogenic mec
Page 52 and 53: 46Ravindran and WilliamsMATERIALS A
Page 54 and 55: 48Ravindran and WilliamsTable I. Mo
Page 56 and 57: 50Ravindran and WilliamsTable IV. D
Page 58 and 59: 52Ravindran and WilliamsACKNOWLEDGE
Page 60 and 61: 54 Jamali et al.(vaginal discharge,
Page 62 and 63: 56Jamali et al.1 2 3 4 5 6 7 8 9 10
Page 66 and 67: 60 Gupta et al.therefore, the prese
Page 68 and 69: 62 Gupta et al.Table II: Dimorphic
Page 72 and 73: 66 Jayraw and RaoteTable I. Effect
Page 76 and 77: 70 Venkatesh, Ramalingam and Vijayl
Page 84 and 85: 78 Ghosh and GayenDescription of th
Page 86 and 87: 80 Ghosh and Gayen30 µm30 µmCBCBC
Page 88 and 89: 82Manjula and Janardananmicrometres
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84Manjula and JanardananMiracidiumF
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86 Hirani et al.haemoglobin concent
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88Hirani et al.ACKNOWLEDGEMENTSJosh
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90 Gupta and SinghOvary rounded, en
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96 Rajendran and DubeOptiMAL and IC
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specifically show how the results a
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THE INDIAN SOCIETY FOR PARASITOLOGY
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