Gametocyte rich parasite lysate was prepared using lysis buffer containing phosphatase inhibitors (20IlM sodium fluoride, 20llM ~-glycerophosphate, and IOOIlM sodium vanadate). For some experiments, 2mM calcium or 2 mM EGTA was added to the lysis buffer. IOOllg of lysate protein was incubated with PfCDPK4 anti-sera (1:100 ratio) for 12 h at 4°C on an end-to-end shaker. Subsequently, 50 III of protein A+G-Sepharose (Amersham Biosciences) was added to the antibody-protein complex and incubated on an end-to-end shaker for 2 h. The beads were washed with phosphate-buffer saline three times at 4°C and were resuspended in kinase assay buffer that contained phosphatase inhibitors.

mmunoprecipitation of PfCDPK4 from parasite lysates

nzyme-linked immunosorbent assay (ELISA) was performed to detect cytokine secretion from human THP-1 macrophages upon activation with LPS. The ELISAs were performed according to manufacturer's protocol. Briefly, THP-1 macrophages were subjected to various treatments and after appropriate time interval the cell culture supernatants were harvested. The capture antibodies for the individual cytokines were diluted 1:250 in coating buffer (0.1 M Sodium carbonate, pH 9.5) and 100 flL was ali quoted into each well of a 96 well ELISA plate (BD biosciences ). The plates were incubated at 4°C for 16 h following which three washes with 0.05% PBS-Tween-20 were given. Blocking was performed using 200 flL of assay diluent (PBS with 10% FCS) per well for 1 h at room temperature following which 1 00 flL of appropriately diluted standards and samples were added and incubated for 2 h at room temperature. A total of 5 washes with 0.05% PBS-Tween-20 were given and the plates were subsequently incubated with 100 flL of detection antibody and streptavidin-HRP for 1 h at room temperature following which 5 washes were given. 100 flL of tetramethylbenzidine (TMB) substrate was aliquoted into each well and incubated for 15 min at room temperature in dark following which 50 flL of stop solution (2N H2S04) was added to terminate the reaction. The absorbance was read at 450 nm and the cytokine levels in the samples were derived based on the OD45o values obtained with standards of known concentration

Cytokine ELISA

with appropriate antibiotics. Serial dilutions of the toxin, made in 0.2% HSA were added to the cells and incubated for indicated time periods. After incubation, the adherent cells were labeled with 0.25 J!Ci eH] leucine per well and the suspension cells with 0.75 J!Ci eH] leucine per well for 3 hat 37 °C. The cells were harvested on to filtermats using an automatic cell harvester, and the incorporation of the eH] leucine in the newly synthesized proteins was estimated using LKB ~-plate counter. Activity was expressed as percentage of control where no toxin was added to the cells. To check the specificity of chimeric toxins for TFR, 10 J.Lg of anti-TFR antibody (HB21) was added to each well prior to the addition of the fusion protein in the competition experiments.

Cytotoxic activity of restrictocin, its mutants and chimeric toxins was checked on a variety of cell lines. The cellular protein synthesis was assayed in the absence and presence of various concentrations of toxin by measuring eH] leucine incorporation in the newly synthesized proteins. Adherent cell lines namely A431, A549, HeLa, L929 and MCF7 were plated in RPMI 1640 containing 10% FCS at a density of 5x I a:~ cells per well in 96 well culture plates and allowed to adhere for 12 h at 37 °C in the presence of 5% C02• Next day, the medium was replaced with 200 J.LI of leucine free RPMI medium containing 10% serum. The leucine free RPMI containing 1 0% FCS was used for seeding partially adherent cell lines, COL0205 and J774A.1. Cells were allowed to adhere for 12 h and the toxin was added in the same medium. The suspension cell lines, HUT102 and K562 were plated at a density of 5X 103 _cells/well in 80% leucine free RPMI containing 18% complete RPMI and 2% serum immediately before use. The medium used at various stages was supplemented

Assay of Cytotoxicity of Chimeric Toxins

conjugated to fluorescene isothiocyanate ( FITC ) or horse radish peroxidase HRP ) , added. In case of the FITC staining, the cells were finally mounted in a medium containing 0. 1 % para-phenylenediamine PPD and 90 % glycerol in NKH buffer, to retard fading of the fluorescent label. In case of HRP staining, the colour was developed with 0. 05 % DAB ( 3', 3-diaminobenzidine tetrahydrochloride ) in NKH buffer with 0. 002 % hydrogen peroxide. The colour was developed for 15 mins. and the reaction stopped by rinsing in phosphate buffer. The cells were examined under a standard 1 ight microscope for HRP staining or a fluorescent microscope ( for FITC staining ) .

Cells were grown to subconfluence on polylysine coated glass coverslips contained in plastic dishes. After washing with NKH buffer ( 145 mM NaCl, 5 mM KCl, 15 mM Hepes, pH 7. 4 ) , the cells were fixed for 10 mins. in 70 % ethanol at room temperature. The cells were again rinsed extensively in NKH buffer and the appropriate dilution of primary antibody added. Following OIN incubation at 4°c, the cells were washed 3 X with NKH buffer and the appropriate second antibody,

Immunocytochemistry.

phosphoricacidformedisreducedbytheadditionof1-amino2napthol~4-sulphonicacid(ANSA)reagenttoproducethebluecolor.Theactivityofthebluecolorwasreadat680nmagainstreagentblankusingaU.V.Spectrophotometer.Suitablestandardswererunthrougheachbatchofassays.Theenzymeactivitywasexpressedintermsofpgofinorganicphosphorusformedhr'1mg'1protein.

Aftereffluentexposure,thecontrolandeffluentexposedfishtissueswereremovedandplacedinabeakercontainingice-coldSEIbuffer(300mMsucrose,200mMNa2EDTA,50mMimidazole,pH7.23)foranalysisofNa+-K+ATPaseactivity.ThetissueswereimmediatelyfrozeninliquidN2andstoredat-80°Cuntilanalyzed. Thespecificactivitiesofsodium,potassium,magnesiumandcalciumdependentATPaseswereassayedaccordingtothemethodsdescribedbyWatsonandBeamish(1980)and Boeseetal.(1982).AdenosinetriphosphatasecatalysestheconversionofATPandADP.Duringthisconversion,onemolecule ofphosphorusisliberated.ATPaseAdenosinetriphosphate^...^Adenosinediphosphate+PTheinorganicphosphorusliberatedwasassayedaccordingtothemethodofFiskeandSubbarow(1925).Inthismethodtheproteinisprecipitatedwithtrichloroaceticacid.Theproteinfreefiltrateistreatedwithaceticacidmolybdatesolutionandthe

Na+K+ATPase,Mg2+ATPaseandCa2+ATPase

Thereactionproduction,p-nitrophenolinacidphosphatewasmeasuredspectrophotometricallyat415nmagainstreagentblank.Theenzymeactivitywascalculatedfromthestandardcurveandexpressedasmicromolesofp-nitrophenolformedperhourpermilligramprotein.Therateofhydrolysisofp-nitrophenolphophateisproportionaltotheenzymepresentinthetissue.p-nitrophenylphosphate+NaoH—phosphat?-—>p -nitrophenol+phosphateThecolordevelopedinalkalinephosphataseactivitywasreadat410nmagainstreagentblankspectrophotometrically.Theactivityoftheenzymewasexpressedaspmolphenolformedmin'1mg'1protein

ThealkalinephosphataseactivitywasestimatedbythemethodofMorton(1955)usingp-nitrophenylphosphatesocolorlessinsolutionbutuponhydrolysis,thephosphategroupliberatesp-nitrophenylwhichishighlycoloredinalkalinesolution

Alkalinephosphatase(AKP:ortho-phosphoricmonoester-phosphohydrolase;E.C.3.1.3.1)

AcidphosphatasewasassayedfollowingtheprocedureofMorton(1955).Theactivityoftheenzymewasexpressedaspmolphenolformedmin'1mg'1protein.

Acidphosphatase(ACP:acidorthophosphoricphosphohydrolase)(EC3.1.3.2)

LDHisthekeyenzymeinvolvedinglycolysis,andisresponsiblefortheanaerobicconversionofpyruvicacidtolacticacid,theterminalstageintheEmbden-Meyerhofpathway.Theenzymeactivitywasdeterminedinthecontrolandeffluentexposedfishbrain,gill,muscle,liver,heart,kidneyandair-breathingorgansfollowingSrikantanandKrishnamurthi(1955).TheopticaldensitiesweremeasuredinaUVSpectrophotometerusing340 nmfilterandtheresultsare expressedaspmolesofformazanmg'1proteinhr’1

Lactatedehydrogenase(LDH)(L-LactateNADoxidoreductase)(EC1.1.1.27)

Thesupernatant(0.5mlcontaining50mgtissue)wasassayedforSDH.Thereactionwasinitiatedbytheadditionof0.5mlofthesupernatant.Controlsreceived0.5mlsucroseinplaceoftheenzymeextract.Afterincubationfor30minat37°C,thereactionwasstoppedbytheadditionof5mlglacialaceticacidandthederivedformazanwasextractedinto5mloftoluene.Afterkeepingitovernightincold,thecolorwasmeasuredinUV-Spectrophotometerat495mMusingsilicacuvettes.Enzymeactivitieswereexpressedaspmolesofformazanmg'1proteinhr'1

Thisisanimportantenzymeinvolvedinthecitricacidcycle.Thehomogenatesofcontrolandeffluentexposedtissueswerepreparedin0.25MicecoldsucroseusingPotterElvehjemtypeglasshomogenizerandcentrifugedat3000rpmfor15min

Succinicdehydrogenase(SDH)(E.C.1.3.99.1

Theacetylcholineconcentrationinthetissueswasestimatedspectrophotometricallyat540nmbythemethodofHestrin(1949)usingformicacid-acetonemixture(0.15mformicacidacetone,3:17V/V)astheextractionmediumAchconcentrationwascalculatedintermsofnmolAch/mgtissue

Acetylcholine(Ach)

Aftereffluentexposure,thetissuesweredissectedout,weighedandhomogenizedin0.25Msucrose.Thehomogenateswerecentrifugedat10,000rev/minatatemperaturebelow8°C.AcetylcholinesteraseactivityofthesampleswasdeterminedatpH7.0usingafinalhomogenateconcentrationof25mgmf1at10°CwithmMacetylcholineiodideassubstrate and0.001or0.002NsodiumhydroxideastitrantfollowingHestron’smethodasgivenbyMetcalf(1951).ProteindeterminationsforalltheChEanalyseswereconductedonaliquotsofthehomogenatesusingamodificationoftheLowryetal.(1951)method.AchEactivityisexpressedinpmolesofacetylcholinechloridehydrolysedmgtissue'1hr'1

Acetylcholinesterase(AchE

0.89%salinesolutioninaTejElon-glasshomogenizerat4°C.Thehomogenatewascentrifugedat4000rpm(3500xg)at4°Cfor20minutes.Theclearsupernatant(organextract)wasusedforestimationofenzymes

Aftereffluentexposure,thecontrolandexperimentalfisheswerekilledbyhammeringonheadanddissectedimmediately.Excisedbrain,gill,muscle,liver,heart,kidneyandair-breathingorganswereweighed(about20mg)andhomogenizedin2mlof

Collectionoftissues

Enzymologicalanalyses

taining withER Tracker™ Blue White DPX: ER Tracker™ is a dapoxyl dye that specifically stains ER in live cells. Since the dye was not found to retain after fixation, live cell staining was performed when fixation was required before staining with antibodies against the CYP proteins. Once washed, the cells were blocked with 3% NGS prepared in 0.001% Digitonin for 30 min at 4°C. This was followed by washing cells with chilled PBS at centrifugation at 805 x g for 10 min at 4°C. Then the cells were incubated with the appropriate primary antibody prepared in 0.001% digitonin for 1h at 4°C. The unbound primary antibody was washed off with chilled PBS three times by centrifugation at 805 x g for 10 min at 4°C. Cells were then incubated with appropriate secondary antibody again prepared in 0.001 % digitonin for 1h on ice. This was followed by three washes with chilled PBS as before. ER Tracker Blue (1p.M) then added to cells which were incubated on ice for 30mins on ice. The excess dye was washed and the cells viewed under the microscope after mounting on slides with anti-fading mounting media

The protocols for immunostaining cells with different antibodies and dyes were standardized individually for the respective staining procedure: Staining with MitoTracker® Red: MitoTracker® probes diffuse passively across the plasma membrane and accumulate into the active mitochondria. 100]1M stock solutions of MitoTracker Red CMX Ros were prepared in DMSO. Cells were stained at a final concentration of 100nM for 10 min at 37°C, at RT in the dark. The excess unbound dye was washed off and the cells were ready to be viewed under the microscope or processed further for fixation and antibody staining. Staining with antibodies (Table 3.9): Log phase cells were centrifuged at 129 x g for 5 min at RT to remove all dead cells. The live cells were washed 2X with PBS to remove any adherent media and FBS. Fixation was carried out with 2% formaldehyde at RT for 15 min. The fixed cells were washed 2X with PBS by centrifugation at 805 x g at RT for 5 min. They were then blocked using 3% Normal Goat Serum (NGS) prepared in 0.01% Saponin for 30 min at RT. After a wash with PBS, the cells were incubated with appropriate dilution of primary antibody prepared in 0.01% saponin for 1h at RT. The unbound antibody was thoroughly washed off by at least three washes with PBS. Incubation with respective fluorophore conjugated secondary antibody, again diluted in 0.01% saponin, and was carried out for 45 min at RT. The unbound antibody was thoroughly washed off by at least three washes with PBS. The cells were resuspended in a small volume of PBS and mounted on a slide along with anti-fading mounting media (10% glycerol and 0.001% P-phenylenediamine)

Immunocytochemistry

between the 5' and 3' flanking regions using the sites Smai and BamHI to generate pBSK+CYP710C1Hyg

The vector pBlueScript SK+ was used as the backbone to generate the replacement constructs. Standard cloning techniques were used (Sambrook et al., 1989). CYP5122A1 allelic replacement constructs were prepared by inserting ORFs encoding Neomycin or Hygromycin resistance between 0.4 Kb 5' and 0.37 Kb 3' CYP5122A1 flanking regions cloned into the vector pBlueScript SK+, to generate the constructs pBSK+CYP5122A1Neo and pBSK+CYP5122A1Hyg respectively. The following steps were performed. (i) A 416bp 5' flanking sequence of CYP5122A1 was amplified by Hi-fidelity PCR (Table 3.6) and cloned in between unique EcoRV and EcoRI sites of pBSK+ vector. (ii) A 378bp 3' flanking region of CYP5122A1 was amplified using Hi-fidelity PCR and cloned in between BamHI and Xbal sites in pBSK+ with the 5'fragment already cloned in. (iii) ORF for Neomycin resistance was amplified from the vector pXG-GFP+2 and cloned in between the 5' and 3' flanking regions already cloned into pBSK+ vector using the EcoRI and BamHI sites, to generate the construct pBSK+CYP5122A1Neo. (iv) To generate the second replacement construct, hygromycin ORF was amplified from the vector pXG-Hyg (Kindly provided by Dr. Stephen M. Beverley, Washington University) and cloned in between the 5' and 3' flanking regions using the sites Smal and BamHI to generate pBSK+CYP5122A1Hyg. Similarly CYP710C1 allelic replacement constructs were prepared by inserting ORFs encoding Neomycin or Hygromycin resistance between 0.36 Kb 5' and 0.37 Kb 3' CYP710C1 flanking regions cloned into the vector pBlueScript SK+, to generate the constructs pBSK+CYP710C1Neo and pBSK+CYP710C1Hyg respectively. The following steps were performed. (i) A 364 bp 5' flanking sequence of CYP710C1 was amplified by Hi-fidelity PCR (Table 3.7) and cloned in between unique EcoRV and EcoRI sites of pBlueScript SK+ vector. (ii) A 378bp 3 'flanking region of CYP710C1 was amplified using Hi-fidelity PCR and cloned in between BamHI and Xbal sites in pBlueScript SK+ with the 5'fragment already cloned in. (iii) ORF for Neomycin resistance was amplified from the vector pXG-GFP+2 and cloned in between the 5' and 3'flanking region cloned into pBlueScript SK+ vector using the EcoRI and BamHI sites, to generate the construct pBSK+CYP710C1Neo. (iv) To generate the second replacement construct, hygromycin ORF was amplified from the vector pXG-Hyg and cloned in

Generation of allelic replacement constructs for generation of knock outs