Qualitative and quantitative estimation of DNA

Determination of the yield

Procedure for DNA isolation

Reagents required for fungal DNA isolationand p

Effect of temperature on xylanase activity

Nitrate reduction test

Nitrate reduction tes

Motif identification

Last node to spike length(cm)

Flow cytometric analysis of cytokine production (IFN-γ)by intracellular staining

Expression and purification of recombinant proteins

pGEX6P2-BPN (1-993 a.a)

Kinetics of bacterial clearance

Sephadex G-100 Column Chromatography

Statistics

Histopathological studies

Bacterial load in the spleens studied by cfu plating

Protection studies

Mammalian cell culture

Agarose gel electrophoresis

AssayofNAD

Endomorphic ectomorph:

SOMATOTYPING

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.

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

Spectroscopic studies

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

The catalytic activity of recombinant PfCDPK4 (and its mutants), as well as irnrnunoprecipitated PfCDPK4 from parasite lysate, was assayed in a buffer

ssay of Protein Kinase Activity

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)

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-

Intracellular free Ca2+ assay

M Tris-HCI pH 8.8, containing 4% stacking gels in O.I25 M Tris-HCI pH 6.8. The gels were run in SDS-PAGE running buffer at a constant current of 40 rnA Proteins were visualized by staining the gels with Coomassie brilliant blue.

The purified proteins were analysed by SDS-PAGE as described by Laemmli ( 1970). Restrictocin and its mutants were analysed on 12.5% resolving gels in 0.375

SDS-PAGE

Following electrophoretic resolution of total RNA, the gels were blotted on to GeneScreen membrane as described by Maniatis et al., 1982 ) .. The RNA gel to be used for blotting was not stained with ethidium bromide. The blotting was performed in 20 X sse or 20 X SSPE, OIN.

Northern blot.

bands seen in the DNA size marker, were marked with a ball -point pen at the places where small holes had been pierced in the gel earlier ( see above ). Thus it was easy to monitor the size of the fragments showing hybridisation to the probe. The gel was then peeled off and the membrane w~shed in 6 X sse with gentle rocking for 10 minutes to wash away any residual agarose sticking to the membrane. After air drying at room temperature, the membrane was baked at so0e for two hours. The baked filter was stored at room temperature in a dessicator, if not used immediately. The dehydrated gel was restained in water containing 0.5 ug I ml ethidium bromide for 30 minutes and examined on a short wave UV transilluminator to check for the presence of any DNA fragments that escaped blotting. The absence of any residual bands indicated that the transfer was complete.

Restriction fragments of DNA resolved on agarose gel were transferred to nylon membrane ( GeneScreen or GeneScreen Plus by the capillary blotting procedure of Southern ( 1975 ) as described by Maniatis et al., ( 1982 ) . After the completion of electrophoresis, the gel was stained and photographed as described earlier. Position of the various bands obtained in the DNA size marker lane were marked by piercing small holes at the two ends of each band in the gel with a yellow tip. The gel was then denatured, neutralised and blotted essentially as described by Maniatis et al., ( 1982 ) . Locally available coarse absorbent paper was used to make the paper towels of the appropriate size. In case of genomic DNA from mammalian cells, the agarose gel was first treated with 0.25 M HCl for 10 minutes, followed by the rest of the procedure as mentioned above. The transfer buffer was 20 X SSPE in all cases. To prevent the absorption of fluid from the 3 MM paper under the gel directly to the blotting paper atop the nylon membrane, the gel was surrounded with polythene sheets to minimise the direct contact between the blotting paper and the 3 MM paper placed under the gel. The blotting was performed for 18 -24 hours. After the transfer was over, the paper towels and the 3 MM papers on top of the nylon filter were peeled off. The gel along with the attached membrane, was turned over and kept on a clean sheet of 3 MM paper with the gel side up. The position of the gel slots was marked with a ball -point pen. Also, the positions of the

southern blot.

Colony lifts were performed essentially as described by Maniatis et al. , 1982 ) . Recombinant colonies were grown 0/N at 37°C to have well separated colonies. The colonies were overlaid with 80 mm diameter nitrocellulose filter circles BA 85, S & S and after the filter became wet throughout, it was peeled off in a single, smooth motion, avoiding the smearing of the bacterial colonies. The plate was reincubated at 37°C for a few hours to regenerate the colonies. The colonies transferred to the filter were lysed to bind the liberated DNA to the nitrocellulose.

Colony lifts.

Transfer of DNA.

Imrnobilisation of DNA L RNA on~ solid support.

incubations were carried out for I h at RT and each incubation was followed by three washings with PBS containing 0.1% Tween-20 (PBST). Post-blocking, the membranes were incubated with 1:1000 dilution ofMA-813 ascites (for detection ofr-bmZP1), MA-451 ascites (for detection of r-dZP3) or rabbit polyclonal anti-r-rG antibodies (for detection of r-rG), followed by an incubation with 1:5000 dilution of goat anti-mouse or goat anti-rabbit immunoglobulins conjugated to horseradish peroxidase (HRPO) (Pierce) respectively. The blots were developed with 0.6% (w/v) 4-chloro-1-naphthol in 50 mM PBS containing 25% methanol and 0.06% H202• The reaction was stopped by extensive washing with double distilled water

The cells (2 - 4 x 1 06) transfected with plasmid DNA were resuspended in minimum volume of 2X sample buffer (0.0625 M Tris, pH 6.8, 2% SDS, 10% glycerol, 5% P-mercaptoethanol, and 0.001% bromophenol blue). The samples were boiled for 10 min and resolved on a 0.1% SDS-1 0% PAGE (Laemmli, 1970). The expression of recombinant proteins was analyzed by Western Blot. The proteins were electrophoretically transferred to 0.45 J.lm nitrocellulose membrane 0/N at a constant current of 30 rnA (milliampere) in Tris-Giycine buffer (25 mM of Tris-HCl and 200 mM glycine) containing 20% methanol (Towbin et al., 1979). Post-transfer, the membranes were washed once with PBS and non-specific sites were blocked with 3% BSA in PBS for 90 min at RT. All the subsequent

Analysis of expressed recombinant protein by immunoblot

COS-I cells were seeded at a density of 2.5 x 105 cells per well in a 6-well tissue culture plate and transfected with plasmid DNA essentially as described above. After 48 h incubation, cells Were trypsinized and counted in a hemocytometer. Cells ( ~ 1 06) were washed twice with PBS and fixed with 0.4% paraformaldehyde in PBS followed by all washings and incubations with respective primary and secondary antibodies in presence of 0.1% Saponin. Antibody concentrations used were same as in indirect immunofluorescence assay. After the final wash, cells were resuspended in PBS and samples were run on an Elite ESP flow cytometer (Coulter Electronics, Hialeh, FL, USA) and data analyzed using WinMDI (version 2.8) software. Cells stained with just secondary antibody were used to account for the background fluorescence. Cells tranfected with VR 1020 vector and probed with primary antibody were used as negative control.

Analysis of mammalian cells, transfected in vitro with the plasmid DNA, by flow cytometry

To investigate if the expressed protein was membrane bound or cytosolic, cells were fixed in 3. 7% paraformaldehyde followed by all washings and incubations with primary and secondary antibodies either in presence or absence of 0.1% Saponin and processed for indirect immunofluorescence as described above.

Localization of the expressed recombinant protein in COS-1 cells

albumin (BSA) in PBS for 2 hat 4°C. For detection of r-bmZPI, a murine monoclonal antibody (MAb), MA-813, generated against E. coli expressed r-bmZP1 (Govind et al., 2000), was used as the primary antibody. The cells were incubated with 1 :500 dilution of MA-813 ascites fluid for 2 hat 4°C. Cells were washed 5 times with PBS and incubated for 1 h with a 1:800 dilution of goat anti-mouse Ig-fluorescein isothiocyanate (FITC) conjugate (Sigma) at 4°C. After washing with PBS, coverslips with the cells were mounted in glycerol : PBS (9 : 1 ), and examined under an Optiphot fluorescent microscope (Nikon, Chiyoda-Ku, Tokyo, Japan). For detecting r-dZP3, MAb, MA-451 (1 :500 dilution of ascites fluid), generated against porcine ZP3f3 (a homologue of dZP3) and immunlogically cross-reactive with dZP3 (Santhanam et al., 1998) was used. For detecting r-rG, rabbit polyclonal antibodies (1:1000 dilution) against E. coli expressed r-rG, was used as primary antibody. The polyclonal antibody was provided by Dr. Sangeeta Choudhury, Project Associate, Gamete Antigen Laboratory, National Institute of Immunology, New Delhi. Goat anti-mouse immunoglobulins-FITC conjugate (1 :800) and goat anti-rabbit immunoglobulins-FITC conjugate (1 :2000; Pierce) were used for detecting anti-dZP3 and anti-rG antibodies respectively

Initial standardization of transfection conditions was done using VRbmZPl plasmid DNA and COS-I mammalian cell line. In brief, cells were cultured in T-25 tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C with 5% C02. For subculturing, cells were trypsinized (0.5% trypsin + 0.2% EDTA in DMEM without FCS), centrifuged at 250 X g for 10 min, resuspended in DMEM supplemented with 10% FCS and aliquoted into T-25 flasks. For transfection, cells were seeded on coverslips in a 24-well tissue culture plate at a density of 5x 104 cells/well, a day prior to transfection. To standardize in vitro transfection conditions for optimum expression of bmZP1, varying amount of plasmid DNA was mixed with lipofectamine in DMEM devoid ofFCS (final reaction volume 200 f.!l) and incubated at RT for 45 min. The cells on the coverslips were washed twice with plain DMEM devoid of FCS. DNA-Iipofectamine complex was added dropwise to the cells and the plate incubated for 8 h at 3 7°C in humidified atmosphere of 5% C02• Subsequently, 1 ml of DMEM containing 10% FCS was added per well and cells allowed to grow for 48 h. After incubation, cells were processed for visualization of r-bmZPl by indirect immunofluorescence assay. Cells were washed twice with phosphate buffer saline (PBS; 50 mM Phosphate and 150 mM NaCI, pH 7.4), fixed in chilled methanol (-20°C) for 3 min and blocked with 3% bovine serum

Detection of the expressed recombinant protein following i11 vitro transfection of mammalian cells with the plasmid DNA.

6000 X g for 15 min at 4°C. Plasmid DNA was purified from the pellet using QIAGEN DNA purification kit according to the manufacturer's instructions. The purified plasmid DNA (1.5 -2.0 mg/ml) was dissolved in autoclaved double distilled water and stored in aliquots (500 f.!l each) at -20°C until further use.

A single colony of the respective clones was picked up from a freshly streaked LB + Kan (50 f.!g/ml) plate, inoculated into 5 ml of LB + Kan medium and incubated for 8 hat 37°C with vigorous shaking (-250 rpm). Subsequently, 500 fll of this primary culture was inoculated into 500 ml LB+ Kan and grown at 37°C 0/N. The culture was centrifuged at

Purification of plasmid DNA in large amount

N-VITRO TRANSFECTION OF MAMMALIAN CELLS WITH V ARlO US PLASMID DNA CONSTRUCTS AND CHARACTERIZATION OF EXPRESSED RECOMBINANT PROTEINS

ThebloodbicarbonatewasestimatedbythemanometricmethodofVanslykeandCullen(1969).

StandardBicarbonates

After transfection the cells were harvested and RNA was isolated from the celllysates using Trizol reagent (Invitrogen) and purified according to the manufacturer's directions. Briefly, the cells were lysed directly in the culture dish by adding 1ml of Trizol reagent to each well. The homogenized sample was incubated at room temperature for 5 min to permit complete dissociation of the nucleoprotein complexes. For purifying the RNA, 200pl of chloroform was added, the tubes were shaken vigorously for 15 seconds and incubated at room temperature for 2-3 min. Tubes were centrifuged at 12,000 ref for 15 min at 40C. The aqueous phase was collected, mixed with 500pl isopropanol and incubated at room temperature for 10 min. Centrifugation was carried out at 12,000 ref for 10 min at 40C. The supernatant was carefully removed and the RNA pellet was washed with 1ml of 70% ethanol by vortexing and then centrifuging at 7500 ref for 5 min. The pellet was air dried and dissolved in 20pl of NFW.

RNA isolation from celllysates

Procedure:

β- galactosidase assay was performed in a 96 well format. Briefly, 4000-5000 cells were plated in 96 well tissue culture coated plate. Cells were transfected with reporter plasmid after 18 -24 hrs and after 48 hrs the cells were washed once with D-PBS. 50μl of lysis buffer was added to the well and cells were lysed by freezing plate at -70°C and thawing at 37°C. Cells were pipette up and down and then the plate was centrifuged at 9000 X g for 5 minutes. The supernatant from each plate was transferred to clean eppendorf tube. Immediately prior to assay the ONPG cocktail was prepared as below: 47 μl 0.1 M sodium phosphate (pH 7.5)22 μl 4 mg/ml ONPG1 μl 100X Mg solution30μl of each well extract was added to microtitre well plate and70μl of ONPG cocktail was added to each well. The plate was kept on ice throughout the procedure. After addition of ONPG cocktail the plate was transferred to 37°C and the development of colour was monitored every 10 minutes for development of color. After development of yellow colour, the reaction was stopped by addition of 150μl of 1M sodium carbonate to each well

Lysis Buffer: 0.1% Triton X-100/0.1 M Tris-HCl (pH 8.0). 450 ml distilled water 50 ml 1M Tris-HCl (pH 8.0) 0.5 ml Triton X-100 detergent • 100X Mg++ solution: 0.1 M magnesium chloride 4.5 M 2-mercaptoethanol Stored at 4°C. • 0.1 M sodium phosphate (pH 7.5)41 ml 0.2 M Na2HPO4 9 ml 0.2 M Na H2PO4 50 ml distilled water • 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1 M sodium phosphate (pH 7.5) containing 2 mM β-mercaptoethanol, Stored at –20°C. • 0.1 mg/ml β-gal standard: 0.1 mg/ml β-gal in 0.1 M sodium phosphate (pH 7.5) containing 2 mM 2-mercaptoethanol Stored at 4°C. • 1 M sodium carbonate in water

Solutions:

β-gal assay in transfected cells

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions by either CDFD or an outsourced sequencing facility

DNA sequencing

To 2 ml of the fresh overnight culture of the recipient strain grown in Z-broth, 108 pfu of P1 lysate was added and incubated at 37°C without shaking for 15 min to facilitate phage adsorption. The unadsorbed phage particles were removed by centrifugation at 4000 rpm for 5 min and the pellet of bacterial cells was resuspended in 5 ml of LB broth containing 20 mM sodium citrate to prevent further phage adsorption. This was incubated at 37°C for 30 min with slow shaking to allow for phenotypic expression of the antibiotic resistance gene. The mixture was then centrifuged, and the pellet was resuspended in 0.3 ml of citrate buffer. 100 μl aliquots were plated on appropriate antibiotic containing plates supplemented with 2.5 mM sodium citrate. A control tube without the addition of the P1 lysate, was processed in the similar way as described above. In case of selection for nutritional requirements, the infection mixture was centrifuged, washed once in 5 ml of citrate buffer and plated without phenotypic expression

P1 transduction

hydrolysis of the non-fluorescent derivative dichlorodihydrofluorescein. In the presence of an appropriate oxidant, dichlorodihydrofluorescein is oxidised to the highly fluorescent 2, 7 -dichlorofluorescein. Log phase cultures were taken and dead cells pelleted at 129 x g for 5 min at RT. The live cells were resuspended in fresh phenol red-free DMEM containing 10% FBS to get a cell density of 107 cells per mL. The cells were loaded with the dye (Stock solution prepared in DMSO to a final concentration of 1pg/pL) by incubating every 107 cells with 2pL of stock solution for 15 to 20 min on an end to end shaker at RT and then washed with medium. The cells were incubated for another 15 min to allow de-esterification to occur. 200pL was aliquotted into each well of a black plate and a basal reading taken at 485nm/ 520nm. Subsequently stained cells were exposed to appropriate treatments and fluorescence monitored at appropriate intervals of time. For each experiment, measurements were prepared in quadruplets and expressed as arbitrary f1uorescence intensity units (AFU)

CM-H2DCFDA (5-(and-6)-chloromethyl -21,7'-dichlorodihydro fluorescein diacetate, acetyl ester) has been used as a detector of ROS as described previously (Mukherjee et al., 2002).This probe is a non-polar, non-fluorescent dye that diffuses readily into cells, where it is trapped by

Assay for measuring intracellular ROS

Ultra competent cells were prepared by using the method described by Inoue (Inoue et al., 1990). Briefly, the DH5-a cells were grown in the SOB culture medium (20gm/L Tryptone, 5 gm/L Yeast extract, 0.5 gm/L Sodium chloride, 2.5 mM Potassium chloride and 10mM Magnesium chloride, pH 7.0) at 18°C till the O.D.6oo of 0.55 was attained. The flasks were then shifted to ice-water bath for 10 min. The cells were harvested by centrifugation at 3220 x g, all media was discarded and the cell pellet was resuspended in Inoue transformation buffer (55mM Manganese chloride, 15mM Calcium chloride, 250mM Potassium chloride, 10mM PIPES, pH 6. 7). The suspension was centrifuged at 3220 x g, the buffer discarded and cell pellet was resuspended in fresh Inoue transformation buffer. DMSO (1.5mL/20mL of buffer) was added and the cells were frozen at -70°C. Cells were checked for transformation efficiency and were used if transformation efficiency was above 5 X 108 transformed colonies / Jlg of DNA

Preparation of ultra-competent cells of E. coli DHS-a

Total cellular polyphosphates were quantified viapolyacrylamide-Tris borate gel electrophoresis (PAGE-TBE).Briefly, PAGEwas performed with Tris-borate buffer (pH 8.3) todeterminethe quantityand the typeof polyphosphatesextracted fromyeaststrains. Equal amount of total RNA (20 to 100 μg) was loadedon 34%PAGE-TBE gel (22cm long, 16 cm wide and 0.8 mm thick) and electrophoresedat 500Voltsfor 20-24 hin cold-roomtill the marker dye bromophenol blue(BPB)had migrated 15-16 cm awayfrom the well.After electrophoresis, total polyphosphates werevisualizedby staining the gel with 0.05%toluidine blue staining solutionfollowed by destaining. Polyphosphates wereobserved both as asmearin the top most portion of the gel as well asdiscreet bands of long chain polyphosphatesand shortchain polyphosphatesin middle andbottom half of the gel,respectively.Polyphosphateband intensity in the gelwas quantified using ImageJ software (http://rsbweb.nih.gov/ij/)and relative amountsof long chain and short chain polyphosphatesin C. glabratacells werecalculated

Quantitative analysisof polyphosphates

Complementary-DNA synthesis was doneusing reverse transcriptase enzyme and oligo-dT primers. For this, 1 μg good quality RNA was treated with1μl(1 unit) DNase I for 15 min to remove DNA contamination. Next, SuperScript III First-Strand Synthesis System kit (Invitrogen) was used to synthesize cDNA according to the manufacturer’s instructions. cDNA synthesized was stored at -20 ̊C till further use

Synthesis of complementary DNA (cDNA)

A microtipful of cellsfor each yeaststrainfrom appropriate mediumwassuspended in 10μlzymolyase cocktailandincubated at 37ºC for 90 min. 2 μlof zymolyase-treated cell suspension was used as template in 25 μlPCR reaction

Yeast colony PCR

Malachite green reagent

Reaction Buffer

Cell lysis Buffer

Calcineurin phosphatase assay

Phenylmethylsulfonyl fluoride (PMSF)

CHX pulse chase assay was performed as essentially described by Zhou (2004). Cycloheximide (CHX), a protein biosynthesis inhibitor was used to determine the half-life and stability of protein of interest. CHX blocks translation elongation step, thereby halting the synthesis of new proteins and therefore, time course degradation of protein can be studied. Briefly, parental and profilin-stable cells were seeded in 35 mm culture dishes and treated with CHX (50 μg/ml) the following day. Cells were harvested at different time points and level of protein was determined by immunoblotting

Cycloheximide (CHX) chase assay

Overnight grown yeast were sub-cultured at 0.2 OD600and growntill 0.8 OD600. Cells equivalent to 1 OD600were harvested and washed twice with SC-Uramedium to remove any residual uracil from the cells. Cells were incubated in SC-Ura medium containing 3 μCi/mL [14C]uracil for 1, 5, 10 and 20 min, and RNA was extracted as described in Section 2.2.8. Equal total RNA was resolved on a formaldehyde agarosegel and transferred to an N+Hybond membrane (GE Life Sciences). Radiolabeled rRNA was detected using a phosphorimager scanner (Fujifilm FLA-9000)

[14C]uracillabelling of total RNA

Triton X-100, 0.5 mg/ml RNase A, 40 μg/ml propidium iodide). Cell nuclei were then incubated for 30 min. at 30°C and were subsequently analyzed by FACS. The hypodiploid nuclei in the histograms were considered and represented aspercentage of apoptotic cells

Apoptosis was measured by Nicoletti method (Nicoletti et al., 1991). Cells treated with appropriate apoptotic stimuli for the indicated times were harvested by centrifugation at 800g for 5 minutes at room temperature. Cells were washed once with PBS, and then resuspended in hypotonic PI lysis buffer (1% sodium citrate, 0.1%

Apoptosis

media containing 50 μM 2’2’-dipyridyland grown for 24 h at 28°C with continuous shaking at 200 rpm. Cells were harvested by centrifugation at 7000 g for 10 min at 4 °C, washed twice with 50 mM phosphate buffer (pH-7.4), and finally resuspended in phosphate buffer. The bacterial suspension was then diluted with chelex-100 treated PS to get a final OD600of 1.0 and incubated at 28°C for 5 min. Iron transport assay was initated by adding 55FeCl3(American radiolabeled chemicals, Inc., St. Louis, USA,specific activity 10.18 mci/mg) to a final concentration of 0.4 μM into the bacterial suspension. The radiolabelled stock solution was diluted with water and 1M sodium ascorbate for 55Fe3+uptake and 55Fe2+uptake studies, respectively. For uptake of FeCl3bound vibrioferrin, both vibrioferrin (7.6 mM stock) and 55FeCl3were incubated in 1:1 ratio by diluting it appropriately with water and uptake was initiated with a final concentartion of 0.4 μM. To stop the uptake, 200 μl of bacterial cell suspension was layered and immediately centifuged (13000 g; 1 min) through 300 μl of di-butylphthalate and di-octyl phthalate (1:1) mixture. The upper aqueous layer and organic solvent was aspirated, and pellet was resuspended in 100 μl Triton-X-100. The suspension was added to 5 ml scintillation cocktail, and radioactivity count was determined in the 3H channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). As control, both Fe2+and Fe3+uptake assays were performed in presence of proton motive force uncoupler carbonylcyanidep-trifluoromethoxyphenylhydrazone (FCCP; 50 μM), to distinguish between non-specific uptake of readiolabelled Fe by the bacterial cells. However, no significant increase in the incorporation of Fe2+and Fe3+ was observed in presence of FCCP, which indicated that iron uptake by these strains is energy-dependent process

In vitro transport assay was performed by using radiolabelled iron to measure the capacity of Xanthomonas oryzaepv. oryzicola strains to transport 55Fe(II) and 55Fe(III) forms of iron as described previously with slight modifications (Ardon et al., 1997; Velayudhan et al., 2000). For iron uptake asssay, Xocwild-type BXOR1 strain, ∆rpfF mutant and the complemented strain harboring full length rpfFgenewere grown overnight in PS medium. 0.2% of the overnight grown culture was inoculated in fresh P

55Fe uptake assay

(SCR65/ SCR66, SCR63/ SCR64 and SCR61/SCR62, respectively) designed from the neighbourhood region of the deleted gene.Replacement of ΔrpfFdeletion mutant with the point mutant allele (E141A and E161A: Glutamate to Alanine) (rpfF*) was carried out by transforming XocΔrpfFmutant with pbsks suicide vector harbouring full length rpfF* allele. The DNA fragment carrying the rpfF* allele was constructed by overlap PCR as described previously (Ionescu et al., 2013)using two 21 and 28 bp complementary primers for E141A-F/R and E161A-F/R, respectively; harbouring GAA to GCA substitution (Table 2.2). The mutated rpfF* allele was amplified by using the end primers only (SC14 and SC17) and cloned into pbsks vector with HindIII and XhoI restriction sites. The resulting suicide vector (pRR16 and pRR17) was transformed into ΔrpfFmutant and single recombinants were selected on PSA medium with kanamycin and ampicillin. Colonies were screened for integration of rpfF* (E141A or E161A) allele through homologous recombination with the flanking region of deleted rpfF allele

electroporation. Single Kmr recombinants were selected on PSA plate containing kanamycin. Insertion of the pK18mob vector in xssAgene was confirmed with PCR and sequencing. To further confirm the mutation in the siderophore biosynthetic gene, we did siderophore production assay on Peptone-sucrose agar (PSA)-chrome azurol sulfonate (CAS) (Schwyn and Neilands, 1987). PSA-CAS plate assay indicated that the xssA mutnat of Xocwas deficient in production of secreted siderophore.Deletion of the chromosomal rpfG, rpfC andclpgene of the X. oryzaepv. oryzicolawas accomplished by allelic exchange, following homologous recombination, utilizing the suicide vector pK18mobsacB harboring 5’ region and 3’ regions of the gene of interest (Katzen et al., 1999). 5’ and 3’ regions of rpfG and rpfC andclp gene were first amplified from the BXOR1by PCR using primers indicated in Table no. 2.2 and products were ligated together. After restriction digestion of ligated PCR products and the pK18mobsacB vector with appropriate restriction enzymes, they were ligated to get the plasmids pRR9, pRR10 and pRR11, respectively. These plasmids were then transformed into E. coliDH5α cells. The transformed E. colicells were selected on the LB agar plates containing nalidixic acid and kanamycin. The positive colonies carrying vector with correct inserts were further selected by colony PCR. These donor cells carrying pRR9, pRR10 and pRR11 containing 5’ and 3’ regions of the gene of interest were then transformed into electrocompetent BXOR1 wild-typecells. First crossover (single crossover) was achieved by culturing the cell mixture on Nutrient agar(NA) containing rifampicin and kanamycin,after transformation. The second crossover was allowed by passaging the cells with single crossover in nutrient brothmedium and then selecting on PSAplates containing rifampicin and 5% sucrose. BXOR1with deletion of the rpfG,rpfCand clp genes by double crossover was identified by PCR using pri

Two fragments, each approximately 300 bp in length corresponding to 5’and 3’ end of the rpfFgene were amplified using genomic DNA of Xocwild-type strain BXOR1, and cloned in pBSKS vector to obtain pRR7 (Table S1 and S5). pRR8 was obtained after ligation of Kmrcassette (EZ::Tn5TM<Kan-2>; Madison, WI) in the HindIII site of pRR7. The resulting plasmid (pRR8) was introduced into XocBXOR1 strain by electroporation. Doublerecombinants (Kmrand Aps) were screened on PSA plates containing appropriate antibiotics. Deletion of rpfF(76 amino acids) in the ∆rpfF mutant strain was confirmed by PCR and sequencing. For complementation analysis, full lengthrpfF gene was amplifiedfrom genomic DNA of Xoc Wild-typeBXOR1 strain with HindIII and EcoRI restriction sites and cloned into stable broad host range vector pHM1 (Hopkins et al., 1992)downstream to lacZpromoter to obtain pSC9. The pSC9 plasmid harboring the wild-typerpfFallele was introduced into ∆rpfF mutant strain by electroporation.To obtain the insertional nonpolar mutant in the xssA(xanthomonas siderophore synthesis A), a 321 bp internal fragment of the xssAgene containing the XbaI and HindIII sites was cloned inpK18mob suicide vector, in which the lacZpromoter drives the expression of downstream gene (Schäfer et al., 1994; Windgassen et al., 2000)to obtain pRR12. The resulting plasmid (pRR12) was introduced into Xoc BXOR1 strain by

Construction of mutants in X. oryzaepv. oryzicolaand rescue of the mutation

Identification of selected micro-organism

For complementation studies, C. glabrataORFs, CgFTR1(1.22 kb), CgFET3(1.91 kb), CgYFH1(0.53 kb), CgCCW14(0.64 kb), CgMAM3(1.91 kb)andCgHOG1(1.34 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the PGK1promoter intothe XmaIand XhoI, XbaIand XmaI, XmaIand XhoI, BamHIand SalI, XmaIand XhoI, and BamHIand XmaIsites, respectively, in the CEN-ARS containing plasmid pGRB2.2 (pRK74). For over-expression studies, C. glabrataORFs, CgCCC1(0.95 kb), CgYAP5(1.05 kb)andCgMRS4(0.92 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the constitutive promoter PDC1into the BamHI and SalI, XbaIand XmaI, and XmaI and XhoIsites, respectively, in the CEN-ARS containing plasmid obtained from Addgene (Addgene-ID 45323). All clones were verified by PCR and sequencing analysis

Cloning of C. glabrataORFs

For complementation studies, C. glabrataORFs, CgFTR1(1.22 kb), CgFET3(1.91 kb), CgYFH1(0.53 kb), CgCCW14(0.64 kb), CgMAM3(1.91 kb)andCgHOG1(1.34 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the PGK1promoter intothe XmaIand XhoI, XbaIand XmaI, XmaIand XhoI, BamHIand SalI, XmaIand XhoI, and BamHIand XmaIsites, respectively, in the CEN-ARS containing plasmid pGRB2.2 (pRK74). For over-expression studies, C. glabrataORFs, CgCCC1(0.95 kb), CgYAP5(1.05 kb)andCgMRS4(0.92 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the constitutive promoter PDC1into the BamHI and SalI, XbaIand XmaI, and XmaI and XhoIsites, respectively, in the CEN-ARS containing plasmid obtained from Addgene (Addgene-ID 45323). All clones were verified by PCR and sequencing analysis

Cloning of C. glabrataORFs

supernatant was collected and filtered through a 0.45 μm syringe filter unit. Lentiviral particles carrying either non-targeting shRNA or both IP6K2 directed shRNA constructs were used to infect MEFs for 48 h. At the 36thhour, cells were treated with HU for 12 h, followed by recovery for 6 h, and immunofluorescence was carried out as described in 2.2.6.Immunoblotting with an IP6K2 antibody(Table 2.3) was performed to analyse knockdown levels as described in Section 2.2.10

(Sigma-Aldrich SHC016) or shRNA directed against mouse Ip6k2(Sigma-Aldrich, TRCN0000202175 or TRCN0000202065), were co-transfected with VSV-G and VSV-GP encoding plasmids into thePhoenix amphotropic packaging cell line, using Polyfect reagent (Qiagen), and incubated at 37°C and 5% CO2for virion formation. After 48 h, the

IP6K2 knockdown by shRNA-Plasmidsencoding either a non-targeting shRNA

ZDB-ALT-110825-11

foolish imaginations of his heart

ARTÍCULO 2

The 29th section of the scheme submitted to us says : ” The Federal Parliament shall have the power of making laws for the peace, the well-being, and the good government of the Confederate provinces, and in particular in respect of the following matters.” The powers of the Federal Government will be in reality unlimited. The fact of the enumeration of these thirty-seven heads does not in the least restrain the power of the Federal Government from legislating on everything. The exceptions are few. I would ask the Honorable Premier, for instance, whether the Federal Government has not the power to enact that marriage is a civil contract ? He cannot deny it, and I do not believe that that clause will in any way suit Lower Canada. In a matter of divorce, I consider that the power of legislating upon it ought to be vested in the Federal Government ; but as to the passing of a marriage act, we have the authority of the past to convince us that Lower Canada will never be satisfied with what is proposed in the plan of Confederation. On a former occasion, when a member of the Parliament of Canada moved to enact that marriage should be made a civil contract, all the members for Lower Canada voted against the motion, and the whole country was opposed to it. I shall also inquire whether the Federal Government will not have the right to enact that religious corporations shall no longer exist in the country, or that they shall not be allowed to hold real property, except what is absolutely necessary for their lodging accommodation. According to the resolutions which have been submitted to us, the Federal Government would certainly have this right. It has been said that article 15 of the 43rd resolution replies to this objection, but I can see nothing in that article which restricts the right of the Federal Government to legislate on this matter. The 43rd resolution defines the powers of the local governments, and article 15 of that resolution declares that they may make laws respecting ” property and civil rights, excepting those portions thereof assigned to the General Parliament.” That article reserves to the local legislatures nothing relative to religious corporations, and the Federal Government would have full power to decree that those corporations shall not hold immovable property. The supreme power is that which has the right to legislate upon, and regulate the existence of, the corporations in question, and they can only possess civil rights so long as the Government permits them to exist. The same might be said of most of the institutions to which Lower Canada is attached. I am therefore right in saying that, so far as those things which Lower Canada most holds to are concerned, Confederation is in fact a Legislative union, because upon the Federal Government is conferred the right of legislating upon those subjects which Lower Canada holds most dear.

9

but all would agree that most of the other hospitals and asylums of various kinds should more properly be supported by local than by general resources.

The management of all the Penitentiaries and Prisons naturally fell under the scope of the local authorities ; also that of Hospitals, Asylums, Charities, and Eleemosynary institutions. With regard to these, he would merely say that there might be some which could hardly be considered local in their nature ; such, for example, was the Marine Hospital at Quebec, a seaport where there was an enormous trade

The Effect of Education on Quality of Life in Elderly Females with Urinary Incontinence, Refereeing to Jahandidegan Center in Shiraz-Iran, 2011

Urinary Incontinence in Older Adults

Pathophysiology

Where do I start?

What follows is unavoidably abstract and general in the extreme.

Up to an inch-and-a-half of rain was also expected overnight, which could help.

Three people died

Motivo o razón para realizar algo o para adoptar cierta actitud quiero saber la causa de tu enfado.