The CLD-J domain shares ~51 % similarity with the CDPK from Arabidopsis thaliana AtCPK-l. The homology model of CLD-JD was determined using Swiss Model from EMBL. The template model used was CLD-JD of AtCPK-1, which was crystallized as a dimer. The J -domain helices from the two monomers were swapped with each other in this structure (Chandran et aI., 2006). Therefore, the initial homology model generated for the complementary CLD-J domain for PfCDPK4 was also a dimer. To understand the interaction of this helix (Gln358_ Lys371) with CLP of the monomer, this helix was rotated and translated keeping residues 372-375 as the flexible linker region and superimposed on to the helix from the other monomer, which resulted in the initial model for the CLD-J domain monomer. Initially, these flexible linker residues (372-375) were locally minimized using COOT (Emsley and Cowtan, 2004), and the overall structure was refined with slow cooling using annealing of CNS (Brunger et aI., 1998) to remove all the short contacts. Finally, the model quality was checked with the Pro check software (Laskowski et aI., 1996). The homology model was generated with the help of Dr. S. Gaurinath, JNU

Homology Modeling

released from the para-ovarian pad of fat as well as from the peritoneal reflections while care was taken to avoid injury to the ovarian vessels. A ligature was tied around the distal end of the fallopian tube including the ovarian vessels following which the ovary was excised. Hemostasis was secured before the stump of the tube was pushed back into the peritoneal cavity. The peritoneum was closed by continuous sutures using 2-0 silk. The same protocol was followed to perform oophorectomy on the contralateral side. The muscular layer and skin were closed together using surgical clips

The anesthetized mice were operated under strict aseptic conditions inside a laminar flow hood. The mouse was placed over layers of sterile tissue paper and the skin overlying the dorsal flanks was sterilized by wiping with 70% ethanol. The flank was palpated gently to identify the kidney, and an incision(~ 5 mm) was made using a pair of scissors on the overlying skin which penetrated the skin, sub-cutaneous tissue and the muscle layer with the parietal peritoneum being exposed and intact. The para-ovarian pad of fat was identified through the intact peritoneum and a small incision was made on the peritoneum overlying it. The ovarian tissue along with the fallopian tube was mobilized and delivered through the incision site. The ovary was

Bilateral oophorectomy

Hair from the skin overlying the left and right dorsal flanks were removed using electrically operated razor. The skin overlying the abdomen was sterilized by wiping with 70% ethanol. Ketamine (1 00 mg/kg) and xylocaine (2%) (20 mg/kg) were mixed and administered intraperitoneally. The mice were returned to the cage and the onset of anesthetic effect was monitored. The mice were considered to be in surgical anesthesia when there was loss of palpebral reflex, righting reflex, and toe pinch reflex. Respiratory rate and heart rate were monitored continuously.

General anesthesia:

Bilateral oophorectomy, the surgical removal of both the ovaries, was performed in mice to simulate a condition of estrogen depletion. All procedures in mice were performed after obtaining approval from the Institutional Animal Ethics Committee (National Institute of Immunology, New Delhi). Female BALB/c mice were used in the study

Bilateral oophorectomy and sham surgery in mice

Animal experiments

in IOmM Tris/HCI (pH 7.4)/ lmM EDTN 0.5% SDS. The radioactivity associated with the cells was counted in a y-counter (LKB).

Competition binding analysis was performed to compare the affinity of the chimeric toxins with native antibody. Anti-transferrin receptor antibody (HB21) was iodinated using iodogen method as described by Harlow and Lane, (1988). Adherent cell lines, namely, A431 and A549 were plated at 4Xl05 cells per well in a 24 well plate and used 16 h later for the assay. 500 J.Ll medium, same as described in cytotoxicity assay, was used to dispense the cells. HUT102 cells were also plated at the same density in microfuge tubes and used immediately. After 2 washes with binding buffer (0.1% BSA in DMEM), various dilutions of toxin, along with 3 ng of labeled antibody in binding buffer, were added to the cells. The cells were incubated at 25 °C for 2 h with mild shaking, washed three times with binding buffer and lysed

Binding Studies

then estimated by monitoring its competitive binding to the hCG recepto~ in the presence of radiolabelled hCG. The testes homogenates from 10 -14 week old Wistar outbred rats were prepared by the method of Dighe and Moudgal ( 1983 ), and the receptor assay 1975 ), with 1989 ) .

Culture supernate from stable clones secreting alpha hCG was mixed with the culture supernate from stable clones showing J3hCG activity. The mixing was done with shaking at 37°C for 16 hours, to allow the two subunits to associate. The presence of the heterodimer in the culture supernate was

Radioreceptor assay.

EROD

ITALYusinga96wellmicrolitreplatereadbyELISAmicroplatereader(modelEL3/Sx,BioTeKInstrumentsINC).Thefinalsolutionwasreadatawavelengthof450nm.Theplasmacortisolconcentrationwascalculatedbasedonaseriesofstandards.

PlasmacortisolwasmeasuredbyadirectimmunoenzymaticdeterminationofcortisolkitmanufacturedbyEquiparSriviaG.Ferrari,21/N-21047,SARONNO

Plasmacortisol

Thecapabilityofheadkidneyneutrophilstomovewasassayedbyamigration-under-agarosetechniquemodifiedfromNelsonetal.(1975).ThemethodhasbeendescribedbySaloetal.(1998).Thedistance,thecellshadmigratedfromthemarginofthewelltowardsthewellcontainingcasein(directedmigration)andintheopposite direction(randommigration)weremeasuredunderthemicroscope.

Migration

Phagocytesfromtheheadkidneywerestimulatedwithphorbol12-myristate13 -acetate(PMA,SigmachemicalCo)andtherespiratoryburstwasdeterminedbytheluminol-enhancedCLmethod(ScottandKlesius,1981)

Theenzyme-linkedimmunospot(ELISPOT)assaywasusedfortheenumeration of totalimmunoglobulinsecretingcells(ISC)andantigen(BCG)-specificantibody-secretingcells(ASC)inthespleenandtheblood.TheELISPOTassayfollowedby Aaltonenetal.(1994)wasused.

EnumerationofsecretinglymphocyteswithELISPOTassay

TheamountoftotalIgMandspecificantibovineY-globulin(BGG)antibodyinthefishplasmawasmeasuredbyanenzyme-linkedimmunosorbentassay(ELISA)asgiven byAaltonenetal.(1994)

Serumimmunoglobulin

Theheadkidneywasusedasasourceofphagocytesforchemiluminescence (CL)andmigrationassays.Cellsfromthehomogenizedheadkidneywereseparatedwithatwo-stepPercollgradient.Granulocyteswerecollectedatthe1.070-1.090g/cm3interfaceandafterwashingwithrHBSSresuspendedinphenolred-freerRPMl.

Headkidney

Thespleenswereremovedandhomogenisedindividuallythroughanylonnet(80mesh).Forisolationoflymphocytes,thetissuehomogenatewaslayeredonatwo-stepPercolldensitygradientandcentrifugedfor30minat400xg.Thelymphocyteswerecollectedatthe1.040-1.080g/cm3interface,washedtwice(400xg,10min)withrHbss,andresuspendedin2mlrRPMl.Cellswerecountedbytrypanblueexclusioninahaemocytometer(viability>95%)andthenumberoflymphocytesfrombloodandspleenwereadjustedto2x106/ml

Spleen

Abloodsamplewastakenfromthecaudalveinofeachfishwitha1mlheparinisedsyringeand24-gaugeneedle.Thebloodwascentrifuged(400xg,5min)fortheseparationofplasma.Plasmawasstoredfrozen(-70°C)forthedeterminationoftheimmunologicalparameters

Collectionofplasma

Immunologicalanalysis

Results are expressed as mean ±SE. An unpaired two tailed student's t-test using Sigma Plot Version 10 & 11 was used for statistical analys

Statistical analysis

and incubated at RT for 5 min. The O.D. was measured at 595nm spectrophotometrically and quantitated against a standard reference table supplied with the kit. Alternatively, a standard curve was also plotted using different concentrations of Bovine Serum Albumin (BSA) and used to quantitate protein concentration. Electrophoretic separation of proteins: Electrophoresis for protein separation was performed using the Laemmli buffer system (Laemmli, 1970) on 10 or 12% polyacrylamide gels under reducing or denaturing conditions (SDS-PAGE). Polyacrylamide gels were prepared from 30% acrylamide (30% acrylamide, 0.8% bis-acrylamide), 4X running buffer (1.5M Tris-HCl pH 8.8), 4X stacking buffer (O.SM Tris-HCl pH 6.8), 10% SDS and 10% ammonium persulfate (APS). The gels were run in tank buffer (0.025M Tris Base, 0.192M glycine and 0.1% w I v SDS) at 40-60V while samples were in stacking gel and 80-100V once they entered resolving gel. A molecular weight marker (unstained or pre-stained depending on the requirement) containing proteins of known sizes were run to evaluate the approximate molecular weights of the resolved proteins. Visualization of protein bands on Polyacrylamide gels: The protein bands resolved in the gels were stained with Coomassie Brilliant Blue R250 (0.125%w lv CBB R250, 50% v lv methanol, 10% v lv acetic acid) for 15-30 min, followed by washing off excess stain with destain (50%v lv methanol, 10%v lv acetic acid)

SDS-PAGE is used to separate proteins according to their electrophoretic mobility which is a function of the length of the polypeptide chain or molecular weight, in the presence of denaturating agents when the secondary structure is lost. One molecule of SDS binds every 2 amino acids, imparting a net negative charge that is proportional to the length of the polypeptide. When loaded on the gel matrix and placed under an electric field, the negatively charged proteins migrate towards the positively charged electrode and are separated by molecular sieve. Preparation of lysates: After appropriate treatments, cells were harvested and washed 1X in PBS to remove any traces of medium and FBS. The remaining pellet was resuspended in residual buffer. A minimal volume of 2X sample buffer (0.125M Tris HCI pH 6.8, 4% sodium dodecyl sulphate, 20%v /v glycerol, 10% P-mercaptoethanol and 0.01 % bromo-phenol blue) was added drop-wise to the pellet while vortexing to ensure complete lysis. The lysate was denatured by boiling at 99°C for 15 min and debris pelleted down by centrifugation at 10000 x g for 5 min at RT. The supernatant was decanted into a fresh tube and used immediately or stored at -70°C. Estimation of protein concentration in lysates: Total protein concentration of whole celllysates was performed using the CB-X™ Protein Assay kit, by a modification of the Bradford method (Bradford, 1976). In brief, 1mL of chilled (-20°C) precipitant solution was added to 10pL of the whole cell lysate and centrifuged at 10,000 x g for 10 min. The supernatant was discarded and the pellet resuspended in 50pL each of solubilising solution A and B. To this 1mL colour reagent was added, the samples were vortexed

Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis