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  1. Jun 2019
    1. Isolation of inclusion bodies from E. coli cells
    2. Overnight grown primary culture of E. coli cells (1 % v/v final concentration) was inoculated into 1 litre of LB media containing antibiotics. Culture was incubated at 37 oc at 200 rpm. Growth was monitored by measuring absorbance of E. coli broth at 600 nm. Culture was induced by adding 1 mM IPTG at an OD of 0.6 and was harvested after 4 hrs of induction. Samples were taken on an hourly basis after induction to check the kinetics of protein expression. Un-induced and induced E. coli cells were analyzed by SDS-PAGE to check the expression of recombinant protein.
    3. Growth and expression of recombinant proteins in E. coli cells
    4. Primary culture of E. coli was grown in LB medium containing either ampicillin (Amp) and/or kanamycin (Kan) to final concentration of 100 j..tg/ml and 25 J..tg/ml respectively. Depending on the vector construct, antibiotics were used for expression of different proteins as described in Table 3.1. Medium was inoculated with 1 ml glycerol stock of E. coli and incubated overnight at 37 oc at 200 rpm.
    5. Preparation of primary culture of E. coli cells
    1. The DFD between an atom pair is the normalized frequency distribution of the interatomic distances sampled from equal time snapshots taken from the MD simulations. DFDs of corresponding atoms taken from the different simulations were used to qualitatively compare the effect of mutational perturbations on the HbS fiber. Considering that the fiber simulations were carried out only for a relatively short time scale of 1.2ns, the calculated DFDs might suffer from errors due to limited sampling. Hence a quantitative comparison of the different DFDs were not attempted, however given that the global parameters monitored during the simulation had already become reasonably stable after 0.2ns (Chapter!, Table 5), it is expected that the gross features of the DFDs would remain unaltered even in much longer simulations.
    2. Distance Frequency Distribution (DFD)
    3. The fluctuation maps (Fu) were calculated from the MD trajectories of the 0-chains as described earlier (Hery et al, 1997). The fluctuation value Fy is given by the equation, where dy(t) is the distance between a pair of designated atoms ( ca atoms as used here) at time t and the angle brackets represent time averages. The Fy values are the standard deviation of interatomic distance. The fluctuation maps in Figure 6 has a black dot wherever the Fu value is less than or equal to 0.5A. Thus dark regions of the map indicate those parts of the molecule which undergo strongly coupled movements.
    4. luctuation Maps
    5. chains were generated in the same way as for the isolated a-chains. Thus the model consisted of eight polypeptide chains ( 4 a-chains and 4 13-chains) with the central two a and two 13 chains making up the axial contact interface. The simulations of the above fiber models were carried out without explicit solvent using the GROMOS96 vacuum force field as implemented within the GROMACS suite of programs. Models of HbS mutants were generated using the program SCWRL and the mutant fiber models were generated as for the native HbS model. SCWRL replaces only the side chains of desired residues with the best possible rotamer of the mutated amino acids. Thus the initial backbone conformation of the isolated a-chains and the fiber models remained identical in the native HbS and the mutants. The MD simulation protocol for the fiber models consisted of an initial steepest descent energy minimization for 1000 steps followed by full MD simulation for 1.2ns at 300 K. Essential parameters of the simulation like the radius of gyration, root mean squared deviation from the initial structure as well as the kinetic and potential energies are summarized in Chapter I, Table 4. It was observed that all the global indicators of the simulation stabilized to their average values within 0.2ns. Hence data from 0.2ns till the end of the simulations were used for all subsequent analysis
    6. MD simulations on the a-chain of HbS and the mutants were carried out using the GROMACS suite of programs (Lindahl eta!, 2001). The initial structure of the native protein was taken from the high-resolution x-ray crystal· structure (Harrington et a!, 1997) of HbS (PDB entry: 2HBS). The structures of the mutants were generated interactively using INSIGHT-II. The initial model structures were placed in a simulation box of size 42.8 x 31.5 x 42.7 A. The closest distance from any protein atom to the walls of the box was not less than 9 A. The system was then solvated by adding a bath of SPC (Berendson eta!, 1981) waters in such a way that the density of the system was as close to 1 as possible. The overall charge of the system was neutralized by placing suitable counter ions wherever necessary (Chapter 1, Table 4). The resulting system was then energy minimized for 1000 steps using the steepest descent algorithm. This was followed by 0.3ns of position restrained MD during which the solvent and counter ions were allowed to move freely but the protein atoms were harmonically restrained to their initial positions. Finally, normal MD was run for 3ns using the default GROMACS force field. Bond lengths were restrained to their equilibrium values using the LINCS (Hess et a!, 1997) algorithm and a cut-off radius of 0.9nm was used for non-bonded interaction calculation. The temperature of the system was maintained close to 300K by weak coupling to an external temperature bath with a coupling constant of 0.1 ps. The integration time step used throughout the simulation was 1 fs. MD simulations of single mutant a-chains (K 16Q, E23Q and H20Q) as well as the double mutants (K16Q/H20Q, K16Q/E23Q and H20Q/E23Q) were carried out in a similar fashion for 3ns. In order to directly analyze the effects of the mutations on the contact interface, we also carried out MD simulations on a miniature model of the sickle hemoglobin fiber consisting of a complex of two hemoglobin tetramers. In the low salt crystal structure of deoxyhemoglobin S (Harrington et a!, 1997) the asymmetric unit consists of two HbS tetramers that pack as two strands of HbS molecules running parallel to the crystallographic a axis. Axial contacts occur between two tetramers of the same strand and lateral contacts occur between tetramers of different strands. A canonical fiber model was generated by taking one of the two tetramers in the asymmetric unit together with its neighbor translated along the crystallographic a axis [Figure 7 (A, B)]. Fiber models incorporating mutant a
    7. Molecular dynamics (MD) simulatio
    8. Electrostatic potentials were calculated by the Finite Difference Poisson-Boltzmann (FDPB) method using the program MEAD running within the PCE web server (http://bioserv.rpbs.jussieu.fr/PCE) (Miteva et al, 2005; Bashford et al, 1992). Additions of hydrogen atoms as well as assigning of atomic radii and charges were performed automatically within the server. MEAD numerically solves the Poisson-Boltzmann equation to yield the distribution of electrostatic potential on the protein surface. Calculations were performed on one of the native a-chains of the 2HbS crystal structure (Harrington et al, 1997) as well as its SCWRL (Dunbrack et al, 1993) generated mutants. All calculations were performed by setting the internal protein dielectric constant to 4 and the external solvent dielectric constant to 80. The ionic strength parameter was held at 0.1
    9. Electrostatic Potentia
    10. The time kinetics of deoxyhemoglobin polymerization were studied in 1.8M, 1.5 and 1 M potassium phosphate buffer (pH 7.25) respectively as described by Adachi and Asakura (1979a, b) using a Cary 400 spectrophotometer equipped with a Peltier temperature controller. Deoxygenation of the hemoglobin sample was ensured by passing moist gaseous nitrogen over the sample in an airtight cuvette and by addition of sodium dithionite. The polymerization of the resultant deoxyhemoglobin samples was initiated by a temperature jump from 4 to 30 oc within 10 sec and the progress of the reaction was followed by monitoring turbidity changes at 700 nm. The delay time was calculated from the kinetic traces
    11. Kinetics of polymerization
    12. with the plunger of a Hamilton syringe. The tube was centrifuged at room temperature at 14,000 rpm for 30 min. The above process of gel-disruption and centrifugation was repeated twice subsequent to which the oil layer was aspirated and suitable aliquots from the supernatant were taken for estimation of Csat by Drabkin' s reagent (Goldberg eta!, 1977).
    13. he gelation concentrations of HbS constructs were determined by the dextran-Csat method of Bookchin et a! (Bookchin et a!, 1999). This method allows measurement of Csat under near-physiological conditions and at much lower concentration of HbS (about 5-fold or less) than that required in standard Csat assays, but essentially provides the same information. Briefly, a suitable aliquot of a concentrated solution of hemoglobin in potassium phosphate buffer (0.05 M, pH 7.50) was taken in a 1.5 ml micro-centrifuge tube. A concentrated dextran (70 kDa) solution prepared in the same buffer was added to it and mixed well. This mixture was overlaid with 0.5 ml of mineral oil, chilled on ice bath and deoxygenated with an anaerobically prepared dithionite solution through an airtight Hamilton syringe. The final concentrations of dextran and dithionite in the mixture were 120 mg/ml and 0.05 M respectively. The deoxygenated sample above was allowed to polymerize at 37°C for 30 min after which the gel under the oil layer was disrupted
    14. Measurement of gelation concentration, Csat
    15. All experiments were carried out on a Beckman XL-A analytical ultracentrifuge, equipped with absorbance optics, and an An60-Ti rotor, at 20 °C. Sedimentation velocity experiments were performed at 40,000 rpm. Data were collected at 540 nm and at a spacing of 0.005 em with three averages in a continuous scan mode. The protein concentration varied in the range 4-40 IJ.M (heme) in 50 mM phosphate buffer, pH 7.2
    16. Analytical Ultracentrifugation experiments
    17. acid. The respective buffer baselines were subtracted from the sample CD data. The ellipticity of the protein samples is reported as mean residue ellipticity (MRE) in deg/cm2/dmol units. The first derivative UV spectra of the oxy and deoxy-HbS were recorded on a Lambda Bio20 spectrophotometer (Perkin Elmer Life ScieAces). The hemoglobin concentration used for the spectral measurements was approximately 50 )!M on heme basis.The spectra of unliganded proteins was recorded subsequent to deoxygenating the hemoglobin samples by passing moist gaseous nitrogen extensively over the sample in an airtight cuvette. Completion of deoxygenation was ascertained by recording the visible spectrum of the deoxygenated Hb sample.
    18. Circular dichroism (CD) spectra were recorded on a J71 0 Spectropolarimeter (Jasco, Japan) fitted with a Peltier type constant temperature cell holder (PTC-348W). The calibration of the equipment was done with (+)-! 0-camphorsulfonic
    19. Spectroscopic studies
    20. The synthetic peptides were purified by RPHPLC on an Aquapore RP300 column (250 x 7 mm) using a 4-72% linear gradient of solvent B (acetonitrile containing 0.1% TFA) in 130 min at a flow rate of 2 mllmin. Globin chains from respective hemoglobins were separated on a similar column of a smaller dimension (250mm x 4.6 mm) under identical conditions but at a flow rate of0.7 ml/min. Electro spray mass spectrometric analysis was carried out on a VG Platform (Fisons) mass spectrometer. The instrument was usually calibrated with standard horse heart myoglobin or gramicidin S solution. Appropriate amount of each sample was taken in 50% acetonitrile containing I% formic acid and analyzed under the positive ion mode.
    21. nalytical procedures
    22. Purified HbS was digested with carboxypeptidase B (200 mg hemoglobin to 1 mg of the enzyme) for 3 hours in freshly prepared 0.05 M Tris-acetate buffer pH 7.1) at 25°C , followed by passage through a cation-exchange column using Whatman CM52.
    23. Preparation of HbS{des arg 141a
    24. concentrators (Amicon), and subjected to reduction with 0.0.5 M sodium dithionite. For this an appropriate amount of anaerobically prepared dithionite solution was added to the reconstituted Hb and the reaction mixture was quickly passed through a Sephadex G25 gel filtration column (30 em x 1.5 em) equilibrated with 0.05 M Tris HCI (pH 7.4), in order to minimize the duration of contact of dithionite with the protein. The reduced Hb was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and loaded onto a CM52 column (1 Ocm x 1.5cm) equilibrated with the same buffer. A linear gradient of 150 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) was employed to elute the protein from the column
    25. Construction of mutant a globins
    26. Reconstitution of a globin and rf chain into HbS tetramers
    27. The semisynthetic a globin was purified from the mixture by CM52 urea chromatography as explained below. The lyophilized sample was dissolved in 0.005 M phosphate buffer (pH 6.9) containing 8 M urea and 0.05 M 2-mercaptoethanol at a concentration of I 0-15 mg/ml and loaded onto a CM52 column (16 em x 1.5 em) equilibrated with the same buffer. After an initial wash with the same buffer, two linear gradients of(a) 100 ml each of0.005 M to 0.03 M and (b) 100 ml each of0.02 M to 0.05 M phosphate buffer (all buffers contained 8 M urea and 0.05 M 2-mercaptoethanol, and were adjusted to pH 6.9) were employed at a flow rate of 45 ml/h to elute the semisynthetic a globin. The column was finally washed with 0.05 M buffer to elute unreacted a31-141 fragment from the column. The elution profile was monitored at 280 nm. The fractions for semi-synthetic a globin were pooled, extensively dialyzed against 0.1% TF A and lyophilized. The semisynthetic yield of the protein varied between 35% to 45%
    28. Purification oftlte semi-!)yntltetic a globin
    29. V8 protease-mediated semisynthesis of a globin was carried out at 4°C in 0.05 M ammonium acetate buffer (pH 6) containing 30% 1-propanol. For this, the lyophilized samples of natural or synthetic analogs of a 1-30 and respective a31-141 were individually prepared in water. Suitable volumes of the complementary fragments were mixed to obtain a 1:1 molar ratio and lyophilized. The lyophilized material was dissolved in appropriate amount of ammonium acetate buffer (pH 6). To this solution, a suitable volume of 1-propanol was added to a final concentration of 30% 1-propanol and 20 mg/ml substrate. The mixture was cooled on ice subsequent to which suitable volume of V8 protease solution prepared in water ( 1% w/w of substrate) was added. The ligation reaction mixture was incubated at 4°C for 24 hours. The extent of synthesis was monitored on RPHPLC by loading an aliquot of the reaction mixture on an analytical reverse phase column. The reaction was stopped by addition of chilled 5% TF A solution (0.2 fold v/v) and lyophilized
    30. Construction of mutant a globins
    31. Peptides were synthesized by standard solid phase synthesis protocols using Fmoc chemistry on a semi-automated peptide synthesizer (Model 90, Advanced Chemtech). For this, Wang resin pre-loaded with N-a-Fmoc-Glu was used as the starting material. The stepwise coupling of Fmoc amino acids was performed with DIPCDIIHOBT activation procedure. The coupling of each step was monitored by Kaiser test for free amine and wherever necessary, a double coupling was used to increase the yield. Before each coupling step and on completion of the synthesis, the N-terminal Fmoc group was removed using 20% piperidine (v/v in DMF). The peptides were cleaved from the resin and the side chains deprotected with appropriate volume of a mixture containing TF A, ethanedithiol, phenol, thioanisole and water (80:5:5:5:5, v/v). The resin was removed by filtration and the crude cleaved peptides were precipitated using cold diethyl ether and extracted in water. The peptides were purified by RPHPLC and their chemical identity was checked by mass spectrometry
    32. ynthesis of al-30 analogs
    33. The complementary segments of a globin needed for the semisynthesis of mutant chains were prepared by V8 protease digestion (Sivaram eta/, 2001). The a globin was dissolved in 0.01 M ammonium acetate buffer (pH 4) at a concentration of 1.0 mg/ml and digested at 37°C with V8 protease (1: 200, w/w) for 3 hours. The completion of digestion was ascertained by RPHPLC, after which the reaction was quenched by addition of neat TFA to a final concentration of 0.1 %. The complementary segments, al-30 and a31-141, from the digestion mixture were isolated in pure form by size-exclusion chromatography on a Sephadex G50 column (98cm x 2.8cm). The column was equilibrated and run in 0.1% TFA. The lyophilized sample of the digest was dissolved in the above solvent and loaded on to the column. The column was run at a flow rate of 30 mllhour and the elution profile monitored at 280 nm. The individual chromatographic profile of a globin digest showed only two peaks, a31-141 and a1-30 respectively, as expected from a single cleavage at the 30-31 peptide bond. The peak fractions were pooled separately and lyophilized
    34. Generation of complementary fragments, al-30 and a31-141, from heme-free a globin
    35. The a-PMB chain was subjected to acid-acetone treatment to separate the heme from the a globin. Briefly, a solution of concentrated a-PMB chain (5 ml; 30 mg/ml) was added dropwise to I 00 ml of thoroughly chilled acid-acetone solution (0.5% v/v HCI in acetone) with constant shaking, and then incubated at -20°C for 30 min to allow complete precipitation of the globin. The precipitated globin was isolated by centrifugation at 7000 rpm (4°C) for 15 min and the supernatant containing soluble heme was discarded
    36. Preparation of heme-free a chain
    37. The [3-PMB and a-PMB chains were eluted with a linear gradient of 500 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) at a flow rate of 50 ml/hour. The chains were separately concentrated using Centriprep concentrators (Amicon) and stored in liquid nitrogen till further use
    38. The heme bound a and ~ subunits were obtained as described by Bucci (1981 ). Briefly, hemoglobin was reacted with PMB in an eight fold molar excess (8 moles of PMB per mole of hemoglobin). The reaction mixture was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and then loaded onto a CM52 column (30cm x !Scm) that was pre-equilibrated with the same buffer.
    39. Separation of the a and f3 subunits of hemoglobin
    40. Blood was drawn from appropriate source into heparinised tubes. The blood sample was centrifuged at 4000 rpm for 15 min ( 4 °C). The supernatant was discarded, and the erythrocytes (pellet) were subsequently washed thrice with chilled isotonic buffer [0.01 M PBS (pH 7.4)] by centrifugation at 4000 rpm and 4°C for 15 min. The washed erythrocytes were lysed in water. The resultant red cell lysate was then dialyzed extensively against PBS (pH 7.4) at 4°C to obtain stripped hemoglobin (hemoglobin devoid of bound allosteric modulators like BPG). The stripped hemoglobin was then loaded onto a pre-equilibrated DE52 column (30cm x 15cm) after extensive dialysis against 0.05 M tris acetate buffer (pH 8.5). The protein was eluted from the column employing a linear gradient of 500 ml each of 0.05 M tris acetate (pH 8.5) and 0.05 M tris acetate (pH 7) at a flow rate of 50 ml/hour. The purified hemoglobin was estimated spectrophotometrically at 540 nm (molar extinction coefficient= 53236 cm-1/M) and stored at -70°C till further use.
    41. Purification of hemoglobin from bloo
    42. Methods
    43. Fmoc protected amino acids and other chemicals used in peptide synthesis were obtained from Novabiochem (Switzerland). V8 protease and TF A were procured from Pierce Chemical Company (USA), while }-propanol, PMB," Hemin, Dithiothreitol, EDT A were obtained from Sigma Chemical Company (USA). DE52 and CM52 ion exchange resins were purchased from Whatman (UK). Sodium diothinite was procured from Fluka (Switzerland) and Catalase from Boehringer Mannheim (Germany). Carboxypeptidase was obtained from Worthington Biochemical Corporation (USA).
    44. Materials
  2. May 2019
    1. 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
    2. Homology Modeling
    3. DAPI 01 ector Labs, USA), and stained parasites were visualized using Zeiss Axioimager fluorescence microscope and the images were processed using Axio Vision software
    4. Thin blood smears of parasite cultures were fixed with chilled methanol for 2 min. After air drying, washing with PBS and permeabilization was done with 0.05 % saponin in 3% BSA/PBS for 15 min, followed by blocking with 3% BSA made in PBS for Ih. Subsequent incubations with primary antibodies were performed for 2h at room temperature or at 4°C overnight. The smears were washed 3x5 times with PBS. The slides were then incubated with appropriate secondary antibodies (labeled either with fluorescein isothiocyanate (FITC) or Texas Red) for 1 hour at I room temperature. The slides were washed again with PBS and air dried in the dark. Smears were mounted in glycerol containing mounting media that contained
    5. mmunofluorescence Assay
    6. 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.
    7. mmunoprecipitation of PfCDPK4 from parasite lysates
    8. temperature for 2h. The nitrocellulose membrane was washed extensively with PBST and developed using chemiluminescence substrate from Pierce (USA).
    9. 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
    10. Western Blot
    11. A 96-well microplate was coated overnight at 4°C with ovalbumin conjugated peptide in 100 mM carbonate buffer, pH 9.5 (2 /J-g/well). The plate was washed 3 times with PBST and blocked with PBS containing 2% BSA (200/J-l/well) at 37°C for 1 h. Serum samples (diluted in PBS) were added in duplicates (50 (/J-lIwell) at different dilutions (1: 1 00, 1: 1000, 1: 10,000) and the plate was incubated at 37°C for 1 h. The plate was washed and incubated with HRP-conjugated appropriate antibody (1: 1 0,000 dilution in PBS containing 2% BSA) at 37°C for 1 h. The plate was washed thoroughly with PBST and freshly prepared TMB substrate (100/J-lIwell) was added and the reaction was stopped with 2 N H2S04 (50 (/J-l/well) and the absorbance at 450 nm was recorded in an ELISA reader
    12. ELISA
    13. A synthetic peptide (KMMTSKDNLNIDIPS) based on the PfCDPK4 sequence was custom synthesized (Peptron Inc.) and conjugated to keyhole limpet hemocyanin via an additional N terminus cysteine residue. It was used to raise polyclonal antisera against PfCDPK4 in rabbit. First immunization was performed using 1 00 ~g of peptide diluted in PBS and mixed 1: 1 v/v with Complete Freund's Adjuvant (CF A). Subsequently, three booster doses of 50 ~g each were given on the 14th, 28t\ 42nd day post first immunization. Blood was collected from animais on 7th, 21 S\ 35th, 49th day. Antibody titers were checked by ELISA using recombinant proteins or ovalbumin conjugated peptides as an antigen. In all cases, pre immune sera from the same rabbit were used as control
    14. Generation of anti-PfCDPK4 antisera
    15. Parasite cultures were distributed in six well plates (2 ml per well) and pharmacological inhibitors were added at desired concentration. Plates were placed in small gas chambers, gassed and immediately returned to 37°C incubator. The lysates were prepared after ~30 min of the addition of inhibitors
    16. Inhibitor Treatment of gametocyte
    17. suspension through a 26 gauge needle. Lysates were cleared by centrifugation at 14,000 g for 30 min at 4°C and supernatant was used for protein estimation using BCA protein estimation kit (Pierce)
    18. P. Jalciparum infected erythrocytes were lysed by the addition of 0.05 % (w/v) saponin to release parasites, followed by a 30 minute incubation on ice. To remove debris and lysed RBCs were washed with cold PBS followed by centrifugation at 8000g. The lysis buffer containing 10 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, and Ix complete protease inhibitor cocktail (Roche Applied Science) was added to the parasite pellet and homogenized by passing the
    19. Preparation of Parasite Cell Lysate
    20. containing 50 mM Tris, pH 7.5, 10 mM magnesium chloride, 1 mM dithiothreitol, and 100 11M p_32p] ATP (6000 Cilmmol) using 6 Ilg of Myelin Basic Protein. Kinase assays were also performed using "syntide-2" a small peptide substrate (PLARTLSV AGLPGKK) custom synthesized by Peptron, South Korea, and has been used as a substrate for plant CDPKs and CaMKs (Harmon et al., 1994; Hashimoto and Soderling, 1987; Yoo and Harmon, 1996). Reactions were performed in the presence of 2 mM calcium chloride or 2 mM EGTA (0 mM Ca2+) for 40 min at 30°C. When MBP was used as the substrate, reactions were stopped by boiling the assay mix for 5 min in Lammeli's buffer followed by SDS-PAGE. Phosphate incorporation was adjudged by autoradiography of SDS-PAGE gels. When Syntide-2 was used as substrate, reactions were stopped by spotting the reaction mix on P81 phosphocellulose paper (Millipore). The paper strips were air dried followed by washing with 75 mM ortho-phosphoric acid. Phosphate incorporation was assessed by scintillation counting of the P81 paper. In PfCDPK4 inhibition assays, peptide inhibitors were preincubated with proteins in a kinase assay buffer at 25°C for 30-60 min prior to the addition of substrate and ATP
    21. The catalytic activity of recombinant PfCDPK4 (and its mutants), as well as irnrnunoprecipitated PfCDPK4 from parasite lysate, was assayed in a buffer
    22. ssay of Protein Kinase Activity
    23. The protein samples were resolved by SDS PAGE using the Laernrnli buffer system (Laernrnli, 1970). The protein sample was denatured by boiling at 100°C for 10 min in Laernrnli's buffer (List I). Resolving gel (10%) was prepared in a minigel (Bio-Rad, USA) system alongwith 3% stacking gel and the electrophoresis was carried out at 120 volts for 125 min. The gel was stained with 0.25% Coomassie blue R staining solution for Ih followed by destaining with successive washes of de staining solution. Staining was avoided when. gel was used for irnrnunoblotting. Details of reagents used for SDS-PAGE are given in List 1.
    24. SDS PAGE
    25. Protein concentrations were detennined using BCA protein estimation kit (Pierce, .. USA). The assay was perfonned according to the instructions provided by the manufacturer. Various dilutions of the sample or BSA were made in appropriate buffer and 200 J.ll of supplied reagent mix (1 :50 ratio) was added to each well in a 96 well plate. The plate was incubated at 37°C for 1 h and the absorbance was measured at 540 nrn.
    26. Protein Estimation
    27. Tris, pH 7.4, 1 mM dithiothreitol, and 10% glycerol. Protein concentration w~s detennined by densitometry analysis of Commassie stained gels. Protein samples were stored at -70°C until further use
    28. To facilitate the expression of recombinant GST-CDPK4 or its mutants, the desired regions of enzyme were PCR amplified using pGEMT-PfCDPK4 as template and PCR primers which possessed overhangs for XhoI and SmaI restriction enzymes (see List II). Often, the PCR products were cloned in TA cloning vector pGE¥T-I easy. Clones in pGEMT-easy vectors were digested with appropriate restriction enzymes to release the inserts. The released inserts were cloned in expression vector pGEX4T-l to facilitate the expression of recombinant proteins. In some cases, the PCR products were digested directly with restriction enzymes and ligated into expression vectors. The plasmid DNA for expression was used to transform E. coli BL21-RIL (Stratagene) strain for the expression of GST-PfCDPK4 and its mutants. Protein expression was induced by overnight incubation of cells with O.lmM IPTG at 18-20°e. Subsequently, cell pellets were suspended in ice cold lysis buffer, contaiJ;1ing 50 mM Tris, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, 1% TritonX-100, and protease inhibitors (lmM phenylmethylsulfonyl fluoride, 10~g/ml leupeptin, 1 O~g/ml pepstatin) and sonication was performed for 6 cycles of one minute each. The resulting cell debris was removed by centrifugation at 20,000g for 40 min at 4°C. Fusion proteins from the cell lysates were affinity-purified using glutathione-sepharose resin (Arnersham). Briefly, after the protein binding, the resin was washed with lysis buffer, and bound proteins were eluted with 50 mM Tris, pH 8.0 with 10 mM glutathione. Finally, purified proteins were dialyzed against 50 mM
    29. xpression and Purification of Recombinant GST (Glutath ion e-S-Transferase) fusion PfCDPK4 and its mutant
    30. All site-directed mutagenesis studies were performed usmg the QuickChange mutagenesis kit (Stratagene) following the manufacturer's instructions. It is a PCR based method for introducing point mutations, replace amino acids and delete or insert single or multiple amino acids into desired plasmid constructs. Primers containing mutations were designed and PCRs were performed using "wildtype" construct as template. The PCR product was' subjected to digestion with DpnI endonuclease, which is specific for methylated DNA. Following DpnJ digestion, the parental DNA template gets cleaved and DNA containing desired,mutation is selected. The residual mutant nicked DNA was transformed in E. coli DH5a competent cells and the resulting plasmids were isolated and sequenced to confirm incorporation of the desired mutations.
    31. ite directed mutagenesi
    32. incubated on ice for 5 min, buffer N3 (350 J.!l) was added to the mixture and the tube was iriverted 4-6 times until mix appeared cloudy. Cell debris was removed by centrifugation at 12000 x g for 10 min and the supernatant was applied to QIAprep spin columns. Columns were centrifuged at 12000 x g for 1 miri and the flow through was discarded and columns were washed using 750 J.!l of 70% ethanol and centrifuged, at 12000 x g for 1 min. Additional centrifugation was performed to remove the residual ethanol. The columns were placed in a 1.5 ml microfuge tube and DNA was eluted with autoclaved water or 1 mM Tris-HCI (PH 8.0).
    33. Plasmid DNA was extracted using commercially available kit (Qiagen, Germany) as per manufacturer's instructions. For a miniprep, bacterial cell pellet from 5ml freshly grown culture were resuspended in 250 III buffer PI containing RNaseA in a microfuge tube, followed by lysis in 250 III of buffer P2. After the tube was
    34. Plasmid DNA Isolation
    35. 5 III of the ligation mix was added to competent cells and mixed gently and the mix was kept on ice for 30 min before giving a heat shock at 42°C for 1 min. The· mixture was incubated on ice for 2 min and 900 III of LB broth was added to each tube. The cells were recovered by centrifugation at 250 rpm at 37°C for 1 h and were plated on LB agar plates containing the appropriate antibiotic(s) and incubated overnight at 37°C
    36. Transformation in E. coli
    37. The ligation reaction consisted of 10 ng of vector, appropriate amount of insert (insert:vector ratio :: 3: 1), 1 x ligation buffer and 1 U of T 4 DNA ligase (NEB, England). The total volume was made up to 10 III with autoclaved water. The ligation reaction mixture was incubated at 16°C for 12 hrs
    38. Ligation
    39. cycles at 94°C for 30 s, 45°C for 30 s, 68°C for 2 min and final extension at 72°C for 10 min (see table 3.1). PCR products were cloned in pGEM-T easy vector (Promega) and the sequence for the cloned PfCDPK4 gene was obtained by automated DNA sequencing
    40. The PCR reaction was carried out usmg Hi-fi Platinum Taq polymerase (Invitrogen) and primers PfCDPK4_F and PfCDPK4_R (see list II) with the following cycling parameters: 94°C for 2 min initial denaturation followed by 3
    41. To obtain PfCDPK4 gene sequence, BLAST search was done usmg either i TgCDPK1 or the published sequence of other CDPKs in the P. Jalciparum genome sequence. An ORF on chromosome 7 exhibited significant sequence homology with other PfCDPKs. Subsequently, PlasmoDB annotation appeared in the public domain and the gene sequence PF07 _0072 matched with the PfCDPK4 sequence! identified by us. For PCR amplification, primers were designed on the basis of' nucleotide sequence of PFb7 _0072. Total RNA from asynchronous P. Jalciparum, cultures was isolated using RNA easy Kit (Qiagen, Germany) and was used to' synthesize cDNA for reverse transcription (RT). Both complimentary and genomic DNA were used as template.
    42. Molecular Cloning of PfCDPK4
    43. Parasites from synchronized cultures were harvested at different time points of growth to obtain ring, trophozoite and schizont stage parasites. RNA was isolated from these stages by using RNAeasy kit (Qiagen) following manufacturer's protocol. The concentration of total RNA was determined by measuring the absorbance at 260 nm. Purity of nucleic acid preparations were determined by calculating OD26onm / OD28onm ratio, a value of near ~ 1.6-1.8 was taken as a standard of purity. To get stage specific cDNA from RNA, reverse transcription was performed using RT-PCR kit (Invitrogen) that contained random hexamers. Subsequently, the gene of interest was amplified using gene specific primers
    44. Isolation of the parasite RNA
    45. or DNA isolationJrom P. Jalciparum, genomic DNA kit from Qiagen (Germany) was used. Isolation was done following manufacturer's instructions. Briefly, infected erythrocytes (5 ml at 10% parasitemia) were centrifuged at 3,000 g for 2 min. The cells were washed once in cold PBS and resuspended in 1 ml. Following which, 10 ilL of 5% saponin (final concentration 0.05%) was added and' mixed gently. After lysis, the mix was immediately centrifuged at 6,000 g for' 5min. Further steps were, carried out according to the manufacturer's instructions to isolate genomic DNA. DNA was quantified by measuring absorbance at 260 nm I using a UV -spectrophotometer
    46. Genomic DNA Isolation from Parasite Culture
    47. For synchronization, mostly ring stage parasites (10 to 12 h post-invasion) wen~ used. The parasite culture was centrifuged at 200g for 5min and the supernatant was discarded. To the pellet, 4 ml of 5% sorbitol was added, mixed gently and incubated for 15min at 37°C. The mix was shaken 2 or 3 times and centrifuged at 200g followed by washing 3 times in complete medium (list I). The culture was then maintained at 5% hematocrit in a 37°C incubator
    48. orbitol-synchronization 0/ P./alciparum
    49. Parasite culture was used to make a thin blood film on a glass slide. After air drying, the thin smear was fixed in methanol for about 30s. A fresh 5 to 10% , giemsa solution was prepared in phosphate buffer (list I). The slide was placed in a I staining jar and the giemsa solution was poured on the slide for 20 min and' subsequently rinsed thoroughly under running tap water. The stained parasites, were then observed under a light microscope using 100X objective.
    50. Giemsa staining o/thin blood smear o/parasite cultures
    51. medium. The culture volume in 75 cm2 culture flasks, was increased to 25 ml from 12 ml. The flasks were kept at 37°C and the medium was prewarmed before use. The flasks were gassed with a mixture of 5% C02, 3% 02 and 92% N2 for a i minimum of 20 seconds at a pressure of around 5 Ib/in2. The culture medium was changed daily without the addition of RBCs. Blood smears were prepared once or twice a week to check the ~tate of the cultures and the presence of gametocytes. Typically, mature gametocytes were observed after 14-17 days
    52. Fresh stock of parasites was thawed for culture as described above. Thin blood smears were made on the fourth day after setting up the culture. When high , parasitemia with "stressed" parasites was observed, culture volume was increased by the addition of medium. At this stage, fresh RBCs were not added to the culture
    53. Gametocyte cultures
    54. . Jalciparum cultures were maintained as described previously (Trager and Jensen, 1976). Briefly, P. Jalciparum strain 3D7 was cultured at 37°C in RPMI " 1640 medium (list I) in 0+ RBCs supplemented with 10% AB+ human serum or : 0.5% Albumax II (complete medium). All media were preheated to 37°C and care was taken to minimize the handling time outside the 37°C incubator. Cultures were gassed with 5% CO2, 3% O2, and 92% N2 for 20 seconds and maintained at 37°C.
    55. Maintenance of P.falciparum cultures
    56. cryopreserved culture vial was obtained from the liquid nitrogen tank, and thawed quickly at 37°C in a water bath. To the vial, O.lv of 12% NaCl was addeq I slowly, dropwise, while shaking the tube gently. Subsequently, 10v of 1.6% NaCl I was added slowly, dropwise while swirling the tube, followed by centrifugation at 200 g at 20°C for 5 min. The supernatant was discarded and 10v of RPMI 1640 complete media was added, followed by centrifugation at 200 g at 20°C for 5 min.' After removal of the supernatant, pelleted parasites were resuspended in complete I media at 0.5% hematocrit. Cultures were gassed with 5% C02, 3% 02, and 92%' N2 and maintained at 37°C
    57. Revival of cryo-preserved Plasmodiumfalciparum cultures
    58. For cryopreservation of P, Jalciparum cultures, mostly ring stage parasites at a high parasitemia were obtained. The parasites were pelleted by centrifugation at 200 g for 5 min with minimum de-acceleration. To the pellet, 1.5 volume of the freezing solution (list I) was added drop-by-drop, while shaking the vial gently; the ad4ition was completed in ~ 1 min. The medium was then transferred into a sterile cryovial, which was stored in ~he liquid nitrogen tank
    59. Cryopreservation of Plasmodiumfalciparum cultures
    60. Human 0+ or AB+ RBC was obtained from a donor and mixed with heparin (50 units/ml of blood) and centrifuged at 500 g for 10 min with minimu1p. de-acceleration. The supernatant was removed carefully and the pelleted RBCS were washed 3 times with RPMI 1640 to remove serum and buffy coat. Equal amount of RPMI 1640 media was added to packed RBC volume to achieve 50% hem~tocrit and stored at 4°C till: further use
    61. Preparation of RBCsfor culture
    62. lasmodium Jalciparum strain 3D7 (MR4, American Type Culture Collection) was i used for all the experiments except where gametocyte rich culture was required. I For generating gametocytes, 3D7 A a variant of 3D7 was used. The parasite was cultured as describerl below:
    63. lasmodiumfalciparum culture
    64. ynthetic peptides used for various studies were custom synthesized by Peptron I (South Korea). Reagents and solution preparations have been indexed in List I
    65. All the reagents stock and working solutions were prepared in milliQ water. The solutions were autoclaved at 121°C (15 psi pressure) for 15 minutes. Most of the , fine chemicals were purchased from Sigma (USA) unless stated otherwise. Anti-PfCDPK4 polyclonal antibody was raised in NZW rabbit, using KLH conjugate~ I 15 amino acids synthetic peptide designed from C-terminal region of PfCDPK4 kinase domain. Phosphoinositides were purchased from Calbiochem (USA)
    1. Enzymatic assays using acyl-peptidyl substrates were set up as follows: 100-120 llmoles of purified ~PL/RNRP protein, 200 l!M valeryl-FT AA-CoA/ valery 1-FT AAlaninal and 2 mM NADPH were incubated at 30°C for 2 hrs. The protein was precipitated with acetonitrile and the reaction was loaded on C 18 RP HPLC column (250 x 4.6 mm, 5l!, phenomenex). The products could be resolved using following gradient: 0 to 48% B in 25 min, 48% B in 40 min and 70% B in 50 min (A-water with 0.1% TF A and B-acetonitrile with 0.1% TF A) at a flow rate 0.6 ml/min. The elution profile was monitored at 220 nm. The identity of peaks obtained was confirmed by TOF-MS and tandem mass spectrometric analysis using ESI-MS (API QSTAR Pulsar i MS/MS, Applied Biosystems).
    2. The enzymatic assays were performed as described for wild type ~Pl. in chapter 2. The standard reaction mixture contained 100 J.lM fatty acyl-CoA (30 J.lM [1-14C] fatty acyl-CoA (55 mCi/mmole,ARC) and 70 J.lM of unlabeled fatty acyl CoA), 2 mM NADPH and 10-20 nmoles protein for l-2 hrs. Lauroyl aldehyde [ l-14C] was obtained enzymatically from [ l-14C] lauric acid (55mCi/mmole,ARC) using the FadD9 protein, and extracted from TLC by using ethyl acetate. Assays were set up using a total of 100 J.lM ('4C labeled + unlabeled) of lauroyl aldehyde in the presence of 2 mM NADPH and 10-20 nmoles protein for 1-2 hrs. The products were extracted twice in 300 J.ll of hexanes and resolved on silica gel 60 F2s4 TLC plates (Merck) using hexanes:ethyl acetate (80:20, v/v) solvent system. The radiolabeled product was detected by using phosphorimager (Fuji BAS500)
    3. nzymatic assays and product characterization
    4. sodium sulphate, filtered and concentrated. The product was reconstituted in methanol and desalted using LH-20 sephadex column. The identity of the aldehyde was confirmed by TOF-MS and tandem mass spectrometric analysis using ESI-MS (API QST AR Pulsar i MSIMS, Applied Biosystems). Purification of peptide was performed using RP-HPLC. The aldehyde could be resolved from other impurities (including traces of alcohol, valeryl-FTAAlaninol) on Cl8 RP-HPLC column (7.8 x 300 mm, 125A, Waters) using a gradient of 0-48% B in 20 mins, 48% B in 40 mins and 90%B in 50 mins (A: water with 0.1% TF A and B: acetonitrile with 0.1% TF A) using a flow rate of 2 mllmin. The elution profile was monitored at 220 nm and the identity of the aldehyde was confirmed by mass spectrometric analysis
    5. The aldehyde valery I-L-Phe-L-Thr-L-Ala-L-Alaninal (valery!-FT AAlaninal) was synthesized by Fmoc-solid phase solid phase chemistry using the Weinreb AM resin (Novabiochem, 0.63 mM/g) and automated peptide synthesizer (Advanced Chemtech. USA). Fmoc protecting groups of amino acids were removed by 20% piperidine in double distilled dimethyl formamide (DMF). A fourfold excess of respective amino acids were preactivated using HoBt (2 equivalents) in DMF and the coupling was catalyzed by diisopropylcarbodiimide (DIPCDI, 2 equivalents). After synthesis resin was dried with dichloromethane/DCM (3 X) and MeOH (3 X). The Thr side chain protecting group (tertiary butyl) was removed by treatment with 60:40, TF A: DCM, twice. The filtrates were discarded and resin was washed with DCM (3 X) and MeOH (3 X). The dried resin was suspended in tetrahydrofuran (3 ml) in a glass reaction flask (25 ml) under nitrogen, swelled with gentle stirring for 1 h, and then cooled to 0 °C. Cleavage of the peptide aldehyde from the resin was performed by adding lithium aluminium hydride (Aldrich. 2 M equivalents dissolved in THF) drop wise for 30 min at 0°C with constant stirring. The reaction was quenched with careful addition of KHS04 (saturated solution) and stirred until the solution reached room temperature. The resin was then filtered off and washed with DCM (3 X) and MeOH (3 X). The filtrate was treated with sodium potassium tartrate (saturated solution) and organic layer was extracted. This organic layer was dried over
    6. Clzemical synthesis and purification of aldehyde valeryl-FTA-Aianina
    7. proteins of interest were pooled and 1 mM TCEP was added. The protein of interest was collected and stored at -80°C for further use after adding 1 mM TCEP.
    8. The mutant proteins were expressed and purified analogous to wild type RaPt protein. Mutant clones pAC36, pAC50 and pAC38 were transformed in BL21 strain of E. coli. Analogous to the wild type RGPL protein the cells harbouring the mutant expression plasmids were cultured at 37°C to an O.D6oonm of 0.6 and uninduced at 30°C for 6-8 hrs. After harvesting, the cells were resuspended in lysis buffer (1 00 mM phosphate pH: 7 .0, I 0% glycerol) and disrupted using french press at 1100 psi pressure. Cell debris was removed by centrifugation at 50,000 g for 40 min at 4°C. 0. 75 ml L.1 of Ni2+ -NT A slurry was added to the supernatant and incubated at 4°C for 1 hr. This suspension was loaded onto a column working under gravity flow. The resin was washed with wash buffer (100 mM phosphate pH: 7.0, 10% glycerol and 5 mM imidazole) till all unbound proteins were removed. The protein was eluted using elution buffers containing increasing concentration of imidazole. Fractions containing the
    9. Expression and purification of RcPL mutant proteins
    10. ~PL mutants were generated using QuickChange site-directed mutagenesis kit (Stratagene). Mutagenesis reactions were performed in accordance with the manufacturer's protocol using pAC28 (wild type ~PL gene fragment cloned in pET28c, section as template. The details of oligonucleotides used for generating the mutant clones are given in table 3.1. Translationally silent restriction sites were engineered in the oligonucleotides whenever possible, in order to facilitate preliminary screening of mutant clones. Mutant clones were screened by restriction endonuclease analysis and confirmed by automated DNA sequencing
    11. Site directed mutagenesis to generate RGPL mutant clones
    12. Methods
    13. QuikChange site directed mutagenesis kit was procured from Stratagene. The protocol used for chemical synthesis of valery 1-FT AA-CoA ( 7) is described in section Solvents and chemicals used for chemical synthesis were procured from Merck and Sigma. Weinreb resin and fmoc-amino acids were procured from Novabiochem. Other materials used in this study have been listed in chapter 2
    14. Alaterials
    1. Optiphot fluorescence microscope, E600W fluorescence microscope and T2000E Confocal microscope Cl were from Nikon (Tokyo, Japan). Multitemp III water bath and EPS 500/400 power supply were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Gyratory water bath shaker was purchased from New Brunswick Scientific Co., Inc (Edison, NJ). Centrivac and Biofuge table top centrifuge were from Heraeus (Allerod, Denmark). f.!-Quant microplate reader was from Bio-tek Instruments Inc. (Winooski, VT). Protean II polyacrylamide gel system and Mini Trans blot system were from Bio-Rad Laboratories (Hercules, CA). Submarine DNA electrophoresis system was procured fro Bangalore Genei (Bangalore, India). Laminar flow hoods were purchased from Kartos Ltd. (New Delhi, India). Eppendorf 581 OR centrifuge was purchased from Eppendorf (Hamburg, Germany). LS50B flourimeter was from Perkin Elmer Biosystems (Norwalk, CT). Fluostar Optima fluorimeter was from BMG labtech (Offenburg, Germany) BD-LSR flow-cytometer was from Bectinson Dickinson Biosciences (San Jose, CA). Peltier Thermal Cycler-200 was purchased from MJ research (Waltham, MA). Doc-It Gel Documentation system was procured from UVP Bio Imaging System Incorporation (Upland, CA)
    2. Instrumentation
    3. Densitometric measurements for quantitation of signals on immunoblots or ethidium bromide stained agarose gels . were performed using a UVP Gel Documentation instrument, and the acquired data was analyzed on Lab Works image analysis and acquisition software (UVP, v. Data from at least 3 experiments were quantitated to arrive at the average value of the signal. All measurements were normalized to internal loading controls. To determine statistical significance, the data was analyzed by Student's T test and the values were expressed as mean±SEM. The values were considered to be significantly different at p<0.05
    4. Densitometry and statistics
    5. DNA was sequenced by the di-deoxy method (10) at the DNA sequencing facility of Department of Biochemistry, University of Delhi, South Campus, New Delhi, India
    6. DNA sequencing
    7. he DNA fragments eluted from the agarose gel were cloned into pGEM-TEasy vector which allows efficient sequencing using the sequencing primers for T7 and SP6 promoters. 3 )lL of eluted DNA (1 )lg/)lL) was ligated with 1 )lL ofpGEM-TEasy vector in the presence of 1 )lL of T4 DNA ligase in a 10 )lL reaction volume. The reaction was allowed to proceed at 4 oc for 16 h following which 8 )lL of the ligation mix was used to transform DH5-a strain of E.coli following standard protocols (9). The transformation mix was spread onto LB-agar plates containing appropriate ampicillin (100 )lg/mL) and the blue-white selection reagent (40 )lLiplate) (Sigma chemical company). The plate was incubated at 37°C for 12 h following which the white colonies were picked up for screening for presence of the gene of interest.
    8. Sub-cloning ofPCR products into pGEM-TEasy vector
    9. To elute DNA from agarose gel, the samples were loaded on a gel (1-1.8%) cast with low melting point agarose (LMP agarose ). The samples were resolved and visualized under UV transilluminator, and the band of interest was excised quickly using a scalpel blade. The volume of gel slice was quantitated by weighing and the DNA eluted using MinElute Gel Extraction kit (Qiagen) as per manufacturer's protocol. Briefly, the gel was solubilized by incubating it with buffer QG at 50°C for 10 min. The solubilized gel was loaded onto binding columns and centrifuged at 12,000 x g for 1 min. The flow-through was discarded and the column was washed once with buffer PE containing ethanol. The DNA bound to the column was eluted using the elution buffer provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260 nm (A26o) and using the following formula: DNA concentration= A260 X 50 X dilution factor.
    10. Elution of DNA from agarose gel
    11. DNA fragments were resolved on 1-2% agarose gel containing 0.5 )lg/mL ethidium bromide in Tris-Acetate-EDTA (TAE) buffer (40 mM Tris-acetate, 2 mM EDT A, pH 8.1 ). The samples were mixed with 6X loading dye containing bromophenol blue, and the samples were resolved by applying a voltage of -5-7 V/cm. The resolved DNA fragments were visualized under ultraviolet illumination and the relative band size was determined by comparison against a DNA ladder with bands of known sizes. When required, images were acquired using a UVP Gel Documentation system.
    12. Agarose gel electrophoresis
    13. 72°C for 45 s - 1 min. A final extension at 68-72°C for 10 min was performed. Relative expression of specific genes in cells subjected to different treatments was determined by semi-quantitative PCR. The optimal number of cycles required for achieving a linear amplification of serially diluted template was determined, which was then used with other samples to quantify the expression of specific genes. The PCR products were resolved on 1-2% agarose gel containing ethidium bromide and visualized under ultraviolet illumination. The specific primers used are shown in Figure 3.1.
    14. Polymerase chain reaction (PCR) was used to amplify specific nucleotide sequences from eDNA derived from human macrophages. The reaction consisted of Gene Forward primer Reverse primer ER-a 51-GTGGGAATGATGAAAGGTGG-31 51-TCCAGAGACTTCAGGGTGCT-31 51-TGAAAAGGAAGGTT AGTGGGAACC-ER-~ 51-TGGTCAGGGACATCATCATGG-31 31 Bcl-2 51-GTGGAGGAGCTCTTCAGGGA-31 51-AGGCACCCAGGGTGATGCCA-3' Mcl-1 51-CGGCAGTCGCTGGAGATTAT-31 51-GTGGTGGTGGTTGGTTA-31 51-TGGAGTGTCCTTTCTGGTCAACAG-Bfl-1 51-AGCTCAAGACTTTGCTCTCCACC-31 31 iN OS 51-GGCCTCGCTCTGGAAAGA-3 I 51-TCCATGCAGACAACCTT-31 51-CTCCTT AATGTCACGCACGATTTC-Actin 51-GTGGGGCGCCCCAGGCACCA-3 I 31 Figure 3.1. The table shows the forward and reverse pnmers designed agamst specific genes used for amplifying products using PCR. an initial denaturation at 94°C for 4 min, followed by 20-30 cycles of denaturation at 94°C for 30 s, annealing at primer specific temperature for 30 s, and extension at 68
    15. Polymerase Chain Reaction
    16. First strand synthesis of mRNA into eDNA was performed using First strand eDNA synthesis kit from Invitrogen following manufacturer's protocol. Briefly, 4 jlg of total RNA was denatured at 65°C for 5 min in the presence of Oligo dT 16 and dNTPs and incubated at 42°C for another 2 min with DTT, MgCb, and RNaseOUT in 10 X reverse transcription buffer. 1 IlL/reaction of the Superscript Reverse Transcriptase enzyme was added to the denatured RNA and incubated at 42°C for 50 min. The enzyme was denatured by heating at 70°C for 15 min. The reaction was completed by a quick high-speed centrifugation and the complementary RNA strand degraded by incubating with RNaseH for 20 min at 37°C. The preparation was stored at -70°C.
    17. First strand synthesis by reverse transcription
    18. X dilution factor X 40) of the obtained RNA was determined by measuring the absorbance at 260 nm (A26o) and 280 nm (A2so).
    19. Total RNA was isolated from cells usmg TRizol reagent following the manufacturer's protocol. Briefly, 2x106 cells were harvested by non-enzymatic cell dissociation buffer and washed once with PBS. The cell pellet was lysed with 1 mL ice-cold TRizol reagent. The lysate was centrifuged at 12,000 x g for 10 min at 4°C to pellet down cellular debris, polysaccharides, and high molecular weight DNA. The supernatant was gently decanted into a fresh microcentrifuge tube and 200 J.tL of chloroform /mL of TRizol was added and the tube was shaken vigorously for 15 s. The mixture was incubated at room temperature for 2-3 min before centrifugation at 12,000 x g for 15 min at 4°C. This resulted in the separation of the mixture into a lower organic phase and an upper aqueous phase. The aqueous phase containing the RNA was gently aspirated and transferred into a fresh microcentrifuge tube and 500 JlL of isopropanol /mL of TRizol reagent was added to precipitate the RNA. The mixture was centrifuged at 12,000 x g for 10 min at 4°C to isolate the RNA as a pellet. The supernatant was discarded and the pellet was washed once with 70% ethanol, centrifuged and the pellet was air-dried and re-dissolved in appropriate quantity of nuclease-free water. The purity (A26o/A2so > 1.8) and concentration (A260
    20. Total RNA isolation
    21. Molecular biology techniques
    22. The cutaneous lesion developed in the L. major infected footpad and tissue from the corresponding region of normal footpad was harvested for histopathological examination. The tissue was fixed in 4% formaldehyde for 24 h following which the tissue was dehydrated by incubating it with ascending concentrations of alcohol (50%, 70%, and 100% ethanol for 1 h each). Subsequently, tissue clearing was performed by incubating with xylene for 1 h following which paraffin embedding was performed. The embedded tissue was cut into multiple sections of 5 J.tm thickness using a microtome. The paraffin sections were then coated on slides and deparaffinization was carried out by treating with xylene. Subsequently, hematoxylin-eosin staining was performed and the slides were visualized under a light microscope.
    23. Histopathological examination of cutaneous leishmaniasis lesion
    24. 5x105 L.major promastigotes were cultured in 5 mL modified DMEM supplemented with 10% FCS. At the end of 5 days of culture, the stationary phase promastigotes were harvested and resuspended in Hanks balanced salt solution at a cell density of 4x107/mL. The cell suspension was aspirated into a 1 mL syringe and 50 J.!L was injected into the footpad of mice. The mice were returned to the cage and fed ab-limitum. The onset and progression of cutaneous lesion was monitored at 2 weekly intervals by observing an increase in the thickness of the footpad
    25. L.major infection in mouse footpad
    26. The mice were returned to a cage and were kept under a 1 OOW bulb light source to prevent hypothermia. Care was taken to ensure that the eyes are kept covered. The respiratory rate and heart rate were monitored till the mice regained complete consciousness. They were fed ab-limitum post-operatively. Metronidazole (20 mg/kg) was added to the drinking water and the mice were fed this medicated water for 5 days post-operatively. On the th post-operative day, the health of the wound was observed and the surgical clips were removed from the skin
    27. ost-operative care
    28. Sham surgery was performed on mice as described above except that the ovary and tubes after being delivered from the incision site were pushed back into the peritoneum in an intact state
    29. Sham surgery:
    30. 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
    31. 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
    32. Bilateral oophorectomy
    33. 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.
    34. General anesthesia:
    35. 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
    36. Bilateral oophorectomy and sham surgery in mice
    37. Animal experiments
    38. resh complete medium was added and the plates were transferred to 37°C for further incubation. The percentage infection was monitored at appropriate time intervals post-infection by staining the cells with Syto Green 11 nucleic acid dye and the parasite nuclei were visualized by fluorescence microscopy
    39. Human THP-1 macrophages were plated at a density of 2X105 cells per well in a 24 well plate and appropriate treatments were given. The stationary phase L.major promastigotes were opsonized with 1% human AB serum in PBS for 5 min at 37°C following which one wash was given with phenol-red free RPMI-1640 medium. The L.major promastigotes were added to the macrophage culture at a macrophage: parasite ratio of 1:10 or 1:50 and incubated for 6 hat 37°C following which the unbound parasites were removed by giving 3 washes with warm RPMI-1640 medium
    40. Leishmania major infection of human macrophages in vitro
    41. 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
    42. Cytokine ELISA
    43. bacteria. The cells were washed once with ice-cold PBS and the uptake of labeled bacteria was analyzed by flow-cytometry
    44. 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
    45. Measurement of phagocytic ability of macrophages
    46. Nitric oxide (NO) generation within the macrophage was detected using the fluorescent NO-sensitive probe DAF-FM diacetate (7). THP-1 macrophages were harvested and resuspended in serum and phenol-red free RPMI-1640 medium and incubated at room temperature for 30 min in the presence of 1 llM DAF-FM diacetate dye. The cells were washed once with fresh medium to remove the excess probe and kinetic fluorescent measurements were performed on a spectrofluorimeter (BMG Fluostar Optima) at an excitation of 480 nm and emission of 520 nm. Time kinetic measurements were performed after appropriate treatment and the values were represented as arbitrary fluorescence units with the comparisons being made against the fluorescence of the control cells. SNAP (S-nitroso-N-acetylpenicillamine), a photoactivatable nitric oxide donor (8) was used as positive control in the assay.
    47. Measurement of intracellular nitric oxide (NO) generation
    48. were represented as arbitrary fluorescence units and comparisons were made against the untrea!ed control samples. Exogenous addition of hydrogen peroxide to cells was used as a positive control for the assay
    49. he generation of reactive oxygen species in macrophages was detected by fluorimetry using the fluorescent dye CM-H2DCFDA, which can detect hydrogen peroxide, hydroxyl radical, peroxyl radical, and peroxynitrite anion (5, 6). To perform the assay, THP-1 macrophages were washed and resuspended in serum and phenol-red free RPMI-1640 medium and incubated at room temperature for 30 min in the presence of CM-H2DCFDA at a final concentration of 1 JiM. Subsequently the cells were washed once with fresh media to remove the excess probe and fluorescence measurements were commenced on a spectrofluorimeter (BMG Fluostar Optima) at an excitation of 480 nm and an emission of 520 nm. Appropriate treatments were initiated and time-kinetic measurements were carried out and the values obtained
    50. Detection of intracellular reactive oxygen species generation
    51. Intracellular Na + measurement was performed using the fluorescent Na + indicator Sodium Green TM tetracetate. THP-1 macrophages were resuspended in phenol-red free RPMI-1640 medium and incubated with Sodium Green™ at a final concentration of 1 JiM for 20 min at room temperature. The cells were washed once with fresh serum-free media to remove excess probe following which kinetic fluorescent measurements were commenced in a spectrofluorimeter (BMG Fluostar Optima) at an excitation of 480 nm and emission of 520 nm. In situ calibration to determine the dissociation constant (Kct) of the dye at 3 7°C was accomplished by using the indicator dye in solutions of precisely known free Na+ concentration in the presence of the pore forming antibiotic gramicidin (10 J,tM). Intracellular Na+ was calculated using the following formula: where, Kct of the dye is 5.7 mM at 37°C, F is the fluorescence of the experimental sample, Fmin is the fluorescence in the absence of Na+ and Fmax is the fluorescence under saturating concentrations ofNa+ in the presence of gramicidin (10 J.i.M)
    52. Assay for intracellular Na +measuremen
    53. at the acidic and basic endpoints of the titrations. Na+ free and HC03-free buffer were prepared as described by Khaled et al.
    54. Intracellular pH measurement was performed using the long-wavelength fluorescent pH indicator carboxy SNARF-1 AM. THP-1 macrophages were resuspended in serum-free and phenol-red free RPMI-1640 medium (106 cells/mL) and incubated at room temperature for 15 min with SNARF-1 AM at a final concentration of 1 f.!M. The cells were washed once in fresh serum-free media and incubated for 20 min for complete de-esterification of intracellular acetoxymethyl esters. In situ calibration ofSNARF-1 AM was performed to determine the pKa of the dye at 3 7°C by using the ionophore Nigericin (1 0 f.!M), which maintains the intracellular pH the same as that of the controlled extracellular medium in a buffer containing high-K+. Appropriate groups were subjected to different treatments and fluorescence measurements were commenced in a spectrofluorimeter (Perkin Elmer, Waltham, MA, USA) followed by kinetic analysis. The pH was calculated from the fluorescence measurements using the following formula: where pKa of carboxy SNARF-1 AM is 7.5 at 37 °C. R is the ratio of fluorescence intensities (F) measured at two emission wavelengths, 580 nm (AI) and 640 nm (A.2), with fixed excitation at 514 nm. The subscripts A and B represent the limiting values
    55. Measurement of intracellular pH
    56. esterification of the dye. Basal fluorescence was measured in a fluorimeter (BMG Fluostar Optima spectrofluorimeter) at an excitation of 480 nm and an emission of 520 nm. Appropriate treatments were initiated and kinetic fluorescence measurements were performed with the temperature being maintained at 3 7°C. At the end of each experiment, a calibration was performed to convert the fluorescence values into absolute calcium concentration using the following formula: where, Kt is the dissociation constant of Ca2+ -Fluo3-AM complex (325 nM), and F represents the fluorescence intensity of cells, Fmax represents the maximum fluorescence (obtained by treating cells with 1 f.!M Ca2+ ionophore A234187 in the presence of 4 mM CaCh), and Fmin corresponds to the minimum fluorescence (obtained by treating cells with 4 mM EGTA)
    57. Cytosolic free Ca2+ was measured using the fluorescent Ca2+ indicator Fluo3-AM. THP-1 macrophages were harvested and resuspended in Kreb's buffer (118 mM NaCl, 25 mM NaHC03, 4.8 mM KCl, 1.2 mM KH2P04, 1.2 mM MgS04, 11 mM glucose, 1.5 mM CaCh.2H20). Fluo3-AM was added at a final concentration of 0.5 J.LM alongwith 1 J.LM Pluronic acid F-127 to aid in dispersal of the dye. The cells were subjected to constant mixing by end-to-end rotation and incubated with the dye for 20 min at room temperature following which the cells were pelleted and resuspended in fresh Kreb' s buffer and incubated for further 15 min to allow complete de-
    58. Intracellular free Ca2+ assay
    59. Sub-cellular fractionation of THP-1 macrophages was performed after lysis with hypotonic buffer. THP-1 macrophages after appropriate treatment were allowed to swell for 10 min in hypotonic buffer (1 0 mM NaCl, 1.5 mM MgCh, 10 mM Tris-HCl, pH 7 .5) followed by homogenization with 50 strokes using a Dounce homogenizer. More than 90% cellular lysis was ensured by visualizing under a light microscope, and immediately after lysis, the mitochondrial membranes were stabilized by addition of 2.5x mitochondrial stabilization buffer (525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl, 2.5 mM EDTA, pH 7.5) to a final concentration of 1x. The homogenate was centrifuged at 1300 x g for 15 min to isolate the nuclear fraction. The post-nuclear fraction was further centrifuged at 17,000 x g for 15 min in an ultracentrifuge (Beckman Optima XL-1 OOK ultracentrifuge) to isolate the mitochondria. The post-mitochondrial supernatant was centrifuged at 100,000 x g for 1 h to obtain the membranous fraction as a pellet and the supernatant obtained was the cytosol. The homogeneity of the obtained fractions was determined by probing for fraction specific proteins by Western blotting
    60. Sub-cellular fractionation