temperature for 2h. The nitrocellulose membrane was washed extensively with PBST and developed using chemiluminescence substrate from Pierce (USA).

The proteins separated by SDS-PAGE were transferred from the gel to· nitrocellulose membrane using a blotting apparatus (Bio-Rad, USA). In brief, after removal of the stacking gel, the resolving gel was placed over nitrocellulose membrane and sandwiched with Whatman 3 mm filter paper in a cassette. The cassette was submerged in transfer buffer and transfer was carried out at 150 rnA for 3h at 4°C. Following the transfer, the membrane was carefully removed from the blotting apparatus and blocked with 3% non-fat dry milk protein for Ih. The membrane was washed thrice with PBST and incubated overnight with the primary antibody at 4°C. Following incubation, the membrane· was washed thrice with PBST and incubated with appropriate HRP-labeled secondary antibody at room

Western Blot

bacteria. The cells were washed once with ice-cold PBS and the uptake of labeled bacteria was analyzed by flow-cytometry

The phagocytic ability of macrophages was determined by monitoring the uptake of Bioparticles® Alexa fluor 488 labeled dead E. coli (Molecular Probes, Eugene, OR). 2X105 THP-1 macrophages were plated per well in a 24 well plate. Alexa fluor 488 labeled dead E. coli particles were opsonized with an opsonizing reagent obtained from Molecular Probes. These opsonizing reagents are derived from purified rabbit polyclonal IgG antibodies that are specific for E.coli. These opsonized bioparticles® were transferred to the macrophage culture at an multiplicity of infection (MOl) of 1:10, i.e., 10 bacteria per macrophage. The plates were briefly centrifuged at 250 x g to allow the bacteria to settle at the bottom of the plate and were then transferred to an incubator maintained at 37°C and 5% C02 in air for 1 h. The culture medium was aspirated to remove excess unbound bacteria and the cells were washed 3x with ice-cold PBS. To eliminate fluorescence from non-phagocytosed bacteria adhering to the macrophage membrane, 0.25 mg/mL Trypan blue was added and incubated for 10 min to quench the fluorescence of extracellula

Measurement of phagocytic ability of macrophages

Cell-free protein synthesis inhibitory activity of restrictocin was assayed as described by Harlow and Lane ( 1988). The frozen rabbit reticulocyte lysate was thawed on ice in the presence of 30 J..LI of haemin solution per 0.5 ml vial. The toxin was diluted in 0.2% RNase free BSA and several concentrations were incubated with 10 J..LI lysate, I mM ATP, 0.2 mM GTP, 75 mM KCI, 2 mM magnesium acetate, 3 mM glucose, 10 mM Tris-HCI, pH 7.6, 4 J..LM amino acid mixture without leucine, 0.16 J..LCi eH] leucine, 1.33 mg/ml creatine phosphokinase, and 2.66 mg/ml creatine phosphate in a reaction volume of 30 J.LI. The reaction was carried out at 30 °C for one hour and terminated by adding 0.25 ml of I N NaOH containing 0.2% H202• The reaction mixture was further incubated for I 0 min. at 37 °C, BSA added to a final concentration of 65 J..Lg/ml, and the proteins were precipitated with 15% trichloroacetic acid. The mixture was left on ice for 30 min. for complete precipitation and harvested onto 26 mm glass fibre filters. The filter discs were placed in a manifold harvester (millipore) and rinsed with chilled 5% TCA, before the addition of reaction contents. The filters were thoroughly washed with chilled acetone and dried at 37 °C, for one hour. The dried filters were immersed in organic scintillation fluid, and counted using a liquid scintillation counter (Packard).

Assay for Inhibition of in Vitro Protein Synthesis

Following transfection with calcium phosphate or lipofectin, the cells were selected for neomycin resistance by using the analogue G418 Geniticin in the culture medium. After 48 ·hours post -transfection, the cells were harvested and replated at a lower density ( 0.5 x 104 cells I 60 mm dish). Culture medium containing G418 was then added to the cells. G418 was used at two concentrations -400 ug I ml and 500 ug 1 ml. The cells were cultured in G418 containing medium for 2 - 3 weeks. During this period, the mock transfected cells and the cells transfected with plasmid lacking neo gene, died and the transformed cells formed colonies. Individual G418 resistant colonies were picked up and propagated as independent clones. The culture supernates from these clones were analysed by RIA for BhCG. The stability of the BhCG secreting clones was assessed by culturing with several passages over a few weeks in media with or without G418.

Isolation of stable clones.

RFFIT is used for detennination of rabies virus neutralizing antibody (RVNA) titers. It is an in vitro cell culture based technique in which foci of virus-infected cells are observed by fluorescent antibody staining. In brief, mouse neuroblastoma (MNA) cells were cultured in T-25 tissue culture flasks in DMEM supplemented with 10% FCS at 35°C in humidified atmosphere of0.5% C02. For subculturing, cells were trypsinized (0.5% trypsin + 0.2% EDT A in DMEM without FCS), centrifuged at 150 X g for 10 min, resuspended in DMEM supplemented with 10% FCS and aliquoted into T -25 flasks. Sera from immunized mice were heat inactivated at 56°C for 30 min and the RVNA titers were determined by RFFIT as described previously (Smith . et al., 1996). Briefly, 100 111 of various dilutions of the reference (Standard Rabies Immune Globulin, Biological Research and Reviews, FDA, Maryland, US) and the test sera were mixed with 100 111 Challenge Virus Strain-11 of rabies virus (containing 50 FFD50) in 8-well tissue culture chamber slides and incubated at 35°C in presence of 0.5% C02 for 90 min. After the incubation period, 0.2 ml of MNA cells ( 1 x 1 05) were added to each well and the slides incubated for 40 h following which these were fixed in chilled acetone and stained with FITC conjugated anti-rabies MAb (Centocor Inc, USA) for 45 min. The slides were washed three times with PBS, mounted in glycerol : PBS (9 : 1 ), and examined under fluorescence microscope (Optiphot, Nikon, Japan). Data was expressed as neutralizing antibody titer that is the reciprocal of the serum dilution resulting in a 50% reduction in the number of the virus infected cells in the presence of the test serum.

RAPID FLUORESCENT FOCUS INHIBITION TEST (RFFIT)

Immediatelyafterisolation,thetissueswereweighedandsubjectedtolipidextractionthatwascarriedoutinduplicateaccordingtoFolchetal.(1957)

Totallipids

Salineextracts(0.89%)oftissueswerepreparedinTeflonglasshomogenizer.ThecarbohydratecontentoftheextractwasdoneaccordingtothemethodofShibkoetal.(1967)

Carbohydrates

TotalproteincontentwasdeterminedbytheFolin-CiocalteaumethodofLowryetal.(1951)asmodifiedbyZakandCohen(1961).Bovinecrystallinealbuminwasusedasa referencestandard

Totalproteins

Aftereffluentexposure,thecontrolandexperimentalfisheswerekilledbyhammeringonheadanddissectedimmediately.Excisedbrain,liver,muscle,gill,kidneyandair-breathingorgans werepooled incoldcondition andusedforbiochemicalestimations.

BiochemicalAnalysis

injection, programmed at 20°C min-1 to 200 °C and held for 10 min, then at 10 °C min-1 to 230 °C, and finally at 5 °C min-1 to 320 °C and held for 5 min. Injection temperature was set at 260 °C. High purity helium was used as carrier gas of 1.0 mL min-1 flow-rate. The spectrophotometers were operated in electron-impact (EI) mode, full scan of 40-550 amu or selected ion monitoring (SIM) was used, the ionization energy was 70 e V. Calibration curve was generated using n-hexane stock solutions of standard ergosterol dilutions (5-300 ng/mL) in duplicates and plotting the peak area versus the concentration (Yang et al., 2009).

Gas chromatography (GC) is a common type of chromatography used in analytic chemistry for separating and analysing compounds that can be vaporised without decomposition, while Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles and thus determining masses for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules. Combined together the technique of GC-MS can be used to identify individual components in a mixture and also quantitate them. The sterol extracts prepared in 3.2.C.14 were dried under nitrogen (N2) gas, resuspended in n-hexane and derivatized with BSTF A (N,O-bis (trimethylsilyl) trifluoroacetamide) containing 1% TMCS (trimethylchlorosilane) at 70 °C for 1 hr. The derivative mixture was dried under N2 (gas) to remove excess BSTFA and subsequently re-dissolved in n-hexane. The column temperature was set at 100°C and held for 5 min for

GC-MS analysis of ergosterol

Leishmania! expression vector pXG-GFP+2 was obtained as a kind gift from Dr. Stephen S. Beverley (Washington University). Full length CYP5122A1 (Ld27) had been amplified by PCR using primers (F27P3/F27P2, Table 3.4) and cloned into pGEM-T Easy vector. The ORF was then excised from pGEMT using Notl and cloned into pXG-GFP+2 vector. The transformants were selected for the insertion of the gene in the correct orientation using restriction digestion. Standard cloning techniques were used (Sambrook et al., 1989).

Generation of pXG-GFP+2-Ld27 (CYP5122Al)