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  2. Jul 2019
    1. Romero was assassinated in 1980

      His views on social and political issues was not taken well and eventually got his assassinated.

    1. In the event of armed attack in Europe on one or more of the Parties to the Treaty by any state or group of states, each of the Parties to the Treaty, in the exercise of its right to individual or collective self-defence in accordance with Article 51

      A "attack one, attack all" mentality

    1. Relative distribution of virulence genes among V. parahaemolyticusfrom Cochin estuary, shrimp farm and sea food
    2. Production of phosphatase
  3. Jun 2019
    1. Post-vaccination declination of Transitional B cell population and increase in memory B cells in HBV positive newborns
    1. 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%
    2. Purification oftlte semi-!)yntltetic a globin
  4. May 2019
    1. Quantitative screening for determination of xylanase in shake flask
    2. Sonicated cells of E. coli having recombinant vector was centrifuged. Supernatant was dispensed into 0.2 % v/v xylan agar plate and incubated for 4 h. The plates were then flooded with Congo red solution (0.2 % w/v) for 30 min and destained with 1M NaCl solution till a clear zone of xylan hydrolysis was visible. The plates were gently shaken on a shaker to accelerate the process of staining/destaining
    3. Qualitative detection of xylanolytic activity by plate assay
    4. DETECTION OF XYLANASE ACTIVITY
    1. 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.
    2. ite directed mutagenesi
    3. 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.
    4. Giemsa staining o/thin blood smear o/parasite cultures
    1. cold PBS followed by incubation with fluorophore labeled secondary antibody at appropriate dilution for 30 min at 4°C. The fluorescence was then visualized under a fluorescence microscope or analyzed by flow cytometry
    2. Immunostaining in Live cells: Immunostaining on live cells was performed by harvesting and resuspending cells in ice-cold PBS. The cells were then incubated with an appropriate dilution of the primary antibody for 1 h at 4 °C following which two washes were given with ice-
    3. Immunostaining in fixed cells: The cells were fixed with 4% formaldehyde for 20 min, following which two washes were given with ice-cold PBS. Permeabilization and blocking were performed simultaneously by incubating the formaldehyde fixed cells in PBS containing 0.1% saponin and 3% normal goat serum for 30 min. The cells were washed once with ice-cold PBS. The permeabilized cells were incubated with the primary antibody at an appropriate dilution for 1 h at room temperature following which three washes with ice-cold PBS was given. These cells were then incubated with fluorophore conjugated secondary antibody (IgG) for 1 hat room temperature following which three washes with ice-cold PBS were given. The nuclei were stained with Hoechst 33342 at a concentration of 1 Jlg/mL for 2 min at room temperature. The staining was then visualized under a Nikon TE2000E fluorescence microscope using appropriate filter blocks. Image acquisition was carried out using a high-resolution Retiga Exi camera (Q-imaging, Surrey, BC, Canada) and subsequent image analysis was performed on Image-Pro Plus software v5.5 (Media Cybernetics, Silver Spring, MD). Alternatively, the fluorescence staining was detected by flow-cytometry (BD-LSR, Beckton Dickinson, NJ, USA) using an air-cooled argon ion laser (488 nm) at appropriate florescence channels. Subsequent data analysis was performed on WinMdi software (Microsoft, v 2.9)
    4. Immunocytochemistry
    1. checks the stereochemical quality of protein structures. The output of the program consists of comprehensive listings of the stereochemical parameters. These files were analyzed to determine if the error in the various stereochemical parameters were within acceptable limits. If any parameter ofthe model was found to be outside the accepted range, they were corrected by adjustment of the concerned residue followed by refinement. The elbow angles of all the Fab structures were calculated using web based applet developed by Standfield et a/ (Stanfield et al., 2006). The buried surface areas (i.e. the area rendered inaccessible to a 1.4 A sphere) were determined using PISA web server (Krissinel and Henr:ck, 2007). CONTACT program of the CCP4 package (Elizabeth Potterton, 2003) was used to determine van der Waals contacts and hydrogen bonds between peptide and Fab. Vander Waals contacts were defined to be present between atoms if they were within 4 A of each other. Hydrogen bonds were assigned for donors and acceptor atoms when the distance between them is less than 3.5 A. In case of hydrogen bonds where the nitrogen atom is the donor, the N-H ... O angle should be greater than 120°. When an oxygen atom is the donor, a cut off value of 90° was used. All Fab-peptide complexes ofmAbs; BBE6.12H3 and 36-65 were compared in terms of various parameters such as elbow angle, peptide and CDR conformation, buried surface area, van der Waal contacts and hydrogen bonding. To compare conformations, RMSD in the position of the Ca as well as all atoms were calculated using SUPERPOSE program of CCP4 package (Krissinel and Henrick, 2004). The CDR conformations in the liganded and unliganded forms were also compared.
    2. Deviations from ideal geometry of the various structures were analyzed using PROCHECK (R. A. Laskowski, 1993) from the CCP4 suite. PROCHECK
    3. Validation, analysis and comparison of models
    1. of the plasmid DNA with 0.5 volume of cold isopropanol. The mixture was kept on ice for 10 min. and centrifuged at 12,000 rpm at 4°C for 15 min. DNA pellet thus obtained was washed with 80% ethanol, dried and dissolved in 50 J.!l TE buffer (pH 8.0). Minipreps were screened by restriction digestion. 5 J.tl of plasmid DNA was incubated with 5 units of appropriate enzyme(s) and 150 units of RNaseT1 for 2 h and the products were analyzed on an agarose gel to identify the positive clones.
    2. 5 ml LB containing 100 J.tg/ml of ampicillin was inoculated with single. bacterial colony picked from the culture plates. The culture was grown for 12 h at 37 °C with vigorous shaking. Cells were harvested from 3 ml of culture by centrifugation at 3000 rpm in a microfuge (Plastocraft) at 4 °C for 15 min. Added 200 J.tl of TEG buffer was added to the cells, and tt.ey were gently resusupended to get a uniform suspension and kept on ice for 5 min. 400 J.tl of freshly prepared alkaline-SDS solution was added to the cell suspension and mixed well by inverting the tubes followed by an incubation on ice for 10 min. Subsequently, 300 J.ll of chilled potassium acetate solution was added and mixed thoroughly by vortexing. The mixture was centrifuged at I 0.000 rpm at 4 °C for 15 min.. The supernatant was collected and phenol-chloroform extraction was performed followed by precipitation
    3. Mini Plas~id Preparation and Screening
    1. Progesterone levels were estimated from sera of bonnet monkeys which were bled biweekly using a radioimmunoassay employing reagents and protocol as prescribed by the W.H.O. Matched Assay Reagent Programme (Sufi et al., 1983). Each sample was run in duplicates. Progesterone was extracted from serum (0.1 ml) by the addition of 2 ml of ice-cold ether in each tube and vortexing for 2 min. The tube was immersed in liquid nitrogen in order to flash freeze the serum phase and the unfrozen ether phase which contained the extracted steroid hormone was decanted into another tube. The ether was allowed to evaporate 0/N and 0.5 ml of steroid assay buffer (0.1 M PBS, pH 7 .3, 0. 1% thiomersal and 0.1% gelatin) was added to the tubes and the tubes were incubated at 40°C for 30 min. Steroid sticking to the walls of the tubes was recovered by vigorous vortexing. 100 J..LI of anti-progesterone Ab (at a dilution giving -50% binding of tritiated p.rogesterone in the absence of unlabelled competing progesterone) was then added to the tubes followed by addition of 0.1 ml of 3H-progesterone ( -10,000 cpm/tube). The mixture was incubated for atleast 16 hrs at 4oc. Unbound progesterone
    2. was separated by addition of 0.2 ml of ice cold assay buffer containing 0.625% activated charcoal and 0.0625% dextran and incubated for 30 min at 4oc. This was followed by centrifugation at 2500 rpm for I 5 min at 4°C. The supernatant was carefully decanted into scintillation vials and 4 ml of scintillation fluid (0.4% 2,5 diphenoxazole; 0.01% POPOP [1-4 bis(5-phenyl-2-oxazolyl)benzene] in sulfur free toluene) was added and counted in a liquid scintillation beta counter (Beckman Instruments, California, USA). The amount of progesterone per ml of serum was calculated from a standard curve with known amounts of progesterone in each assay.
    3. Progesterone Radioimmunoassays
    4. Expression conditions for bZP3 under the polyhedrin promoter were standardized using the Northern blot and Western blot analysis of cells infected with the VI virus. Sf9 cells, seeded at a density of 1.5 million in a 35 mm petridish were allowed to attach for I h at 27oc. The medium was removed and the cells were infected with AcNPV (Autographa californica nuclear polyhedrosis virus) or VI at -10 MOl for 1 h. The infected cells were harvested at different time points from 0-84 h pi. The cells (-2X I o6) were washed with chilled PBS and resuspended in I ml of denaturing solution ( 4 M GITC, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, and 0.1 M BME) followed by addition of 50 Jll of 2 M sodium acetate (pH 4) and 500 Jll water saturated phenol and 1 00 Jll chloroform:isoamyl alcohol ( 49: 1 ). The suspension was mixed thoroughly after the addition of each reagent, vortexed for 1 0 sec and cooled on ice for 15 min. The aqueous and the phenol phases were separated by centrifugation at 12,000 rpm for 20 min in a refrigerated microfuge. The aqueous phase was transferred to a fresh tube and 500 Jll isopropanol was added. RNA was precipitated at -20°C for 1 h, and pelleted at 12,000 rpm for 20 min at 40C. The RNA pellet was dissolved in 300 Jll denaturing solution followed by addition of 300 Jll of isopropanol. RNA was reprecipitated at -2ooc for 1 h, washed with 75% ethanol and the pellet collected by centrifugation at 12,000 rpm in a refrigerated microfuge. RNA was dissolved in 25 Jll of 0.5% SDS by heating at 65°C for 10 min and stored at -700C. RNA was quantitated and 5 Jlg of RNA corresponding to each time point was resolved on a 1.2% agarose formaldehyde gel, transferred to a nylon membrane and probed with 32p labeled bZP3 probe. Cells harvested at different time points from -2X 106 cells 12-84 h pi were pelleted down, washed with 10 mM PBS, pH 7.4, and lysed in reducing buffer and resolved on a 0.1% SDS-10% PAGE as described earlier. The supernatant was concentrated to lOX for loading on the gel.
    5. Expression 'Of bZP3 in BEVS
    6. Double stranded plasmid pBluescript-bZP3 DNA was sequenced using Sanger's dideoxy chain termination method (Sanger et al., 1977) using the Sequenase version 2.0 kit according to the protocols recommended by the manufacturer. Purified plasmid DNA (5 J..Lg) and 2 pM of the sequencing primer was used in the sequencing reaction. Table 2 gives a list of the primers used for sequencing of the bZP3 eDNA clones. bZP3 sequence was confirmed by sequencing three independent clones 401, 403 and 404.
    7. Sequencing of bZP3
    1. minigel alongwith unligated vector to test the ligation. The ligated DNA was used to transform competent ~.coli cells.
    2. Wherever possible, the cloning of DNA fragments was achieved by ligation of compatible sticky ends generated on the vector as well as the insert by digestion with the same enzyme. Self ligation of the linearised vector with compatible sticky ends was minimised by dephosphorylation of the vector DNA using bacterial alkaline phosphatase. The ligation conditions for each batch of T4 DNA ligase were standardised using Hind III generated fragments of lambda DNA as a test sample for sticky end ligation. Routinely, 200 ng of vector DNA was mixed with 2 - 5 fold molar excess of the insert fragment DNA, 2 ul each of the 10 X ligase buffer 500 mM Tris. HCl, pH 7. 5, 100 mM Mgcl2 ) , 10 mM ATP, and 200 mM DTT. The final reaction volume was adjusted to 15 - 2 0 ul with sterile double distilled water, and 0.5 - 1 ul of T4 DNA ligase ( 103 units I ml ) was added. The contents were mixed well and incubated at 13°C for 12 -16 hours. An aliquot of 2 ul was electrophoresed on a
    3. Ligation of DNA fragments.
    4. Intensifying screens were from Kiran X-ray Screens, India. X -ray films were from Agfa -Gevaert, Belgium, Kodak, USA, or Hindustan Photo Films, India. Developer and fixer were from Hindustan Photo Films, India.
    5. Materials for autoradiography.
    1. separately on LB plates containing 100 j...tg/ml ampicillin, 80 j...tg/ml of X-gal and 20 mM of IPTG. The plates were incubated at 37°C for 12 h.
    2. The DH5a strain of E. coli was grown overnight (0/N) in LB at 37°C and subcultured ( 1: 1 OO)in 100 ml of fresh LB. The culture was grown until absorbance at 600 nm (A6oo) reached 0.4. The culture was centrifuged at 2500 X g for 15 min at 4°C. The cell pellet was resuspended in 10 ml of freshly prepared sterile ice cold CaC}z (100 mM) solution and incubated for 30 min on ice. Cells were centrifuged at 1800 X g and the pellet was very gently resuspended in 2 ml of chilled CaCh (100 mM) containing 15% glycerol. Aliquots of 100 111 were dispensed into sterile, chilled 1.5 ml eppendorf tubes and stored at -70°C until further use. For transformation, the ligation products from the above reactions were added separately to a vial each of DH5a competent cells thawed on ice. The contents were gently mixed and incubated on ice for 30 min. The cells were then exposed to heat shock at 42°C for 90 sec and incubated on ice for another 2 min. The transformed cells were grown in 1 ml of LB medium for lh at 37°C with shaking for the expression of the ampicillin resistance marker gene W-lactamase). Aliquots from each transformation were plated
    3. Preparation of competent cells and transformation
    1. Thecapabilityofheadkidneyneutrophilstomovewasassayedbyamigration-under-agarosetechniquemodifiedfromNelsonetal.(1975).ThemethodhasbeendescribedbySaloetal.(1998).Thedistance,thecellshadmigratedfromthemarginofthewelltowardsthewellcontainingcasein(directedmigration)andintheopposite direction(randommigration)weremeasuredunderthemicroscope.
    2. Migration
    3. Thereactionproduction,p-nitrophenolinacidphosphatewasmeasuredspectrophotometricallyat415nmagainstreagentblank.Theenzymeactivitywascalculatedfromthestandardcurveandexpressedasmicromolesofp-nitrophenolformedperhourpermilligramprotein.Therateofhydrolysisofp-nitrophenolphophateisproportionaltotheenzymepresentinthetissue.p-nitrophenylphosphate+NaoH—phosphat?-—>p -nitrophenol+phosphateThecolordevelopedinalkalinephosphataseactivitywasreadat410nmagainstreagentblankspectrophotometrically.Theactivityoftheenzymewasexpressedaspmolphenolformedmin'1mg'1protein
    4. ThealkalinephosphataseactivitywasestimatedbythemethodofMorton(1955)usingp-nitrophenylphosphatesocolorlessinsolutionbutuponhydrolysis,thephosphategroupliberatesp-nitrophenylwhichishighlycoloredinalkalinesolution
    5. Alkalinephosphatase(AKP:ortho-phosphoricmonoester-phosphohydrolase;E.C.3.1.3.1)
    6. SamplesofC.punctataweretakenfromtheacclimationtanksandslightlyblottedonpapertowelstoremoveexcesswater.TheywereplacedindividuallyonaWhatmanfilterpaperinaonelitreglassbeaker.Theindividualswereimmediatelytransferredtobigglassdesiccatorscontaining250mlofthefollowingsolutionsfordesiredhumiditylevelsusinggradedsolutionsofKOHasdescribedbySolomon(1951).Watergiving95to97%relativehumidity(RH),mean95%;sodiumchloridegiving72to76%relativehumidity,mean75%;calciumchloridegiving28to31%relativehumidity,mean35%.Thedesiccatorswereplacedinanincubatorataconstanttemperatureof28°±1°Cwithdeterminationsofsurvivalandbodyweightatregularintervals.Thecontainerswereweighedatintervalstothenearest0.1mgandweightlosswasassumedtoequalwater loss
    7. Survivaloutofwater
    1. dried. These were counted directly to determine the total counts. In duplicate tubes, 1pl of the diluted probe was added to 100pg of carrier nucleic acid (tRNA or Herring Sperm DNA) in a total volume of 100pl. To this 500pl of ice-cold 5% TCA was added, mixed thoroughly and incubated on ice for 15-20 min. Glass fiber filters were wet (in duplicate) properly with 5% TCA and then these samples were applied on to them under vacuum. The filters were washed twice with 5ml of chilled 5% TCA and then air dried after rinsing with 2m1 of acetone. All the dry filters were inserted into scintillation vials containing scintillation fluid and the counts were taken in a liquid scintillation a-counter (LKB Wallac, 1219 Rackbeta, Sweden). The percentage incorporation, specific activity and the total amount of RNA made was then calculated according to the standard procedures. % incorporation =Incorporated cpm X100 Totalcpm Total RNA made (ng) = % incorporation X 338 Specific activity of probe = Total cpm incorporated p.g of RNA synthesized
    2. To determine the percentage of incorporation and probe specific activity, 1:10 dilution of the labeled probe was made in NFW. lpl of this was spotted on to duplicate glass fiber filters (Whatman GF/ A, U.S.A.) and ai
    3. richloro acetic acid (TCA) precipitation:
    1. For adherent cells from which lysates have to be prepared , culture medium was removed and cells were washed with ice cold 1X PBS twice and then scraped with cell scraper in Cell Lysis buffer. Cells were rotated at 4°C for 30min at cold room and centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected and protein concentration was estimated using BCA assay. For standard western, 50-70μg of protein was loaded on to the gel
    2. Lysate Preparation for Immuno- blotting
    1. d. 3 μl of sample prepared in low stringency buffer was added to the spot activated with low stringency buffer and incubated in humid chamber for 30 min. and removed using whatman strips. (same protocol was repeated for the samples prepared in high stringency buffer on spots activated with high stringency buffer). e. Stringent washings were given to each spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH7.0 for 30 sec and removed using Whatman filter paper strips. f. 1-2 μl of SAP matrix was added to each spot and allowed to dry. g. The chip was then placed in the SELDI machine
    2. One set of cell extracts was prepared in low stringency buffer by mixing cell extracts and low stringency buffet in 1:1 ratio and another in high stringency buffer. b. 10 μl of low stringency/high stringency buffer was added to the spots on the chip and incubated in a humid chamber for 5 min. c. Buffer was removed using Whatman strips without touching the spot surface. This step was repeated once
    3. Activation of LSAX (strong anion exchange ) array
    4. Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol
    5. TBF method for preparation of high competency cells
    6. For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments
    7. Calcium chloride method
    8. Transformation protocols
    9. Antibiotics were used at the following final concentrations (μg/ml): Rich media Minimal media Ampicillin (for plasmids) 100 50 Ampicillin (chromosome) 30 30 Chloramphenicol (for plasmids) 50 25 Chloramphenicol (chromosome) 25 25 Kanamycin 50 25 Nalidixic acid 50 - Rifampicin 100 - Streptomycin 50 100 Streptomycin 100 200 Spectinomycin 50 100 Tetracycline 15 8 Trimethoprim (for plasmids) 60 30 Chloramphenicol 0.1mg/ml The 10 mg/ml chloramphenicol stock in ethanol was used to make 0.1mg/ml solution in water
    10. Antibiotics
    1. were then centrifuged at 1065 x g for 5 min at 4°C. The pellet obtained was resuspended in 1mL ice cold PBS and washed 5 times with PBS. Finally the pellet was resuspended in 1mL lysis buffer (10mM Tris-HCl containing 0.1% Triton X-100) and fluorescence was measured at 380nm/525nm. To normalize different samples and account for any errors in cell number between samples, 10011L of the above sample was also used for protein estimation, carried out as described above
    2. Monodansylcadaverine (MDC) is a selective marker for autophagic vacuoles (Biederbick et al., 1995). It is an auto-fluorescent drug accumulates in acidic compartments by ion trapping and also is thought to interact with the membrane lipids of the vacuoles. Stock MDC (50mm, prepared in acetic acid) was diluted to a concentration of 50!lM in M199 medium containing 10%FBS. 107 parasites after appropriate treatment were resuspended in 1mL of working stock and incubated in the dark for 10 min at RT. These
    3. Assay for measuring the level of autophagy in Leishmania parasites (MDC staining)
    4. DNA fragments were resolved on 1-2 % agarose gel containing 0.5~-tg/mL ethidium bromide in Tris-Acetate-EDTA (TAE) buffer (40mM Iris-acetate, 2mM EDTA, pH 8.1). The samples were mixed with equal volume of 2X loading dye containing bromophenol blue, and the samples resolved by applying a voltage of 5-7 V /em. The resolved DNA fragments were visualized under ultraviolet illumination (312nm) and the relative band size was determined by comparison against DNA ladder with bands of known sizes. When required the images were acquired using a UVP Gel Documentation system
    5. Agarose gel electrophoresis
    1. 5% glycerol)containing (i) 5′-end-labeled DNAfragmentof 1200 cpm radioactive count(ii) 1 μg each of bovine serum albumin andpoly(dIdC)(iii) the protein at the indicated monomer concentrations and (iv) when required,co-effectorsat specified concentrations. The reaction mixture was incubated at room temperature for 30-minsand the complexes were resolved by electrophoresis on a non-denaturing 5%polyacrylamide gel (39:1 acrylamide:bisacrylamide)in 0.5X TBE buffer pH8.3, at 12.5V/cm for 3 hrs at 18°C.The gels were then dried on a gel drier at 80°C for 45 minsand the radioactive bands were visualised with a Fujifilm FLA-9000 scanner.For DNA bending EMSA, co-effectors were not added in the binding reaction but at aconcentration of 0.1 mM in both the gel and running buffer
    2. The DNA templates were obtained by PCR from E. coligenomic DNA. After 5-end labeling, the PCR fragments were purified by electroelution following electrophoresis on 6% native polyacrylamide gels (Sambrook and Russell,2001). EMSA reactions were performed in 20 μl reaction volume inEMSA binding buffer(10 mM Tris-Cl at pH 7.5, 1 mM EDTA, 50 mM NaCl, 5 mM dithiothreitol, and
    3. Electrophoretic mobility shift assay (EMSA)
    4. Typically 200-300 ng of DNA was used in each ligation reaction. The ratio of vector toinsert was maintained between 1:3 to 1:5 for cohesive end ligation and 1:1 for blunt endligation. The reaction was generally performed in 10 μl volume containing ligationbuffer (provided by the manufacturer) and 0.05 Weiss unit of T4-DNA ligase, at 16ºCfor 14-to 16-hrs. On using the rapid ligation kitfrom Fermentas, incubation was at 22ºC for 1-2 hrs
    5. Ligation of DNA
    6. purification of DNA fragments werefrom Qiagen or HiMedia. The oligonucleotide primers used in this study were mainly synthesised by Ocimum Biosolutions or MWG Biotech. The radioactive chemicals were procured from BRIT Mumbai
    7. Chemicals were obtained from commercial sources. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were mostly from HiMedia laboratories. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4-DNA ligase, DNA-polymerases and DNA size markers were obtained from companies including New England Biolabs, MBI Fermentas and Stratagene.RNA isolation chemicals like Reverse transcriptase, trizol, RNA loading buffers and dyes and RNA size markers were obtained from Invitrogen and Sigma. Protein markers were obtained from MBI Fermentas. Kits for plasmid isolation,
    8. Chemicals
    1. Log-phasecells grown in YPD medium containing or lacking CaCl2and FK506 were collected, PBS-washed and loaded with ratiometric, high affinity, membrane-permeable calcium indicator, Fura-2 AM (10 M; Sigma #47989). After 30 min incubation at 30◦C, labelled cells were washed thrice with cold PBS, suspended in PBS and fluorescence was recorded at 505 nm with dual excitation at 340 and 380 nm. The ratio of fluorescence intensities between 340 and 380 nm, representing Ca-bound and Ca-free Fura-2 molecules, respectively, reflected free intracellular calcium concentrations
    2. Measurement of intracellular calciumlevels
    3. the disrupted gene, BLAST N of the sequences from rescued plasmids was performedagainstC. glabrataGenolevures database (http://www.genolevures.org/blast.html
    4. Identification of disrupted locus in Tn7insertion mutants was carried out as described previously(Kaur et al.,2004). Disrupted locus in each mutant is physically marked with a mini Tn7 transposon derivative containing conditional origin of replication R6K(facilitates Tn7recovery), S. cerevisiae URA3and Klebsiella pneumoniae hphgene (confers resistance to hygromycin B)(Castaño et al.,2003). Briefly,genomic DNA was isolated fromovernight grown Tn7insertion mutants using spheroplast lysis method. After RNAse treatment, 10μgDNA was either digested withMfe1or SpeIrestriction enzymeas the Tn7 cassette lacks these enzyme sites. Followingovernight digestion, DNA wasprecipitated with 1ml ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNApellet was washed twicewith ice-cold 70% ethanol, air driedand resuspendedin sterile MQ water. DNA was recircularized withT4 DNA ligase. Resultant circular plasmid contains the Tn7cassette flanked on either side by the gene,it has disruptedin the genome of C. glabrata.This circular plasmid DNA was transformedin E.coliBW23473 strain,which contains protein Π (the product of the pir gene)required by R6Korifor replication.Transformation of circularized DNA in E.coliBW23473 electrocompetent cells was performedas described below.Plasmids fromselected transformants were isolated and sequenced with outward primers from Tn7right and left ends to sequencethe disrupted gene fragment.For identification of
    5. Tn7insertion mutant rescueand gene identification
    6. E. coli DH5α ultra-competent cells were transformed with plasmid DNA by heat shock at 42 ̊C for 90 sec as described previously in Molecular Cloning-A Laboratory Manual (Sambrook and Russell,2001). Bacterial transformants were selected on LB agarmediumcontaining appropriate antibiotics. Transformants obtainedwere colony purified on LB plates containing antibiotics.Presence of the desired insertwas first verified by colony PCR followed by PCRusing extracted plasmid DNA as template
    7. Bacterial transformation
  5. sg.inflibnet.ac.in sg.inflibnet.ac.in
    1. activated TLC silicagel-60plate and transferred to theTLC chamber. After the solvent had migratedupwards (1.5 cm fromthetop), TLC plate was removed, air dried behind perspex shield, wrapped with cling plastic wrap and was exposed to phophorimager screenfor 2 h. Phosphorimager screen was scanned usingaFugi-FLA 9000 scanner
    2. To resolvephospholipids,a TLC chamber was prepared by pouring50 ml developing solution and sealing the chamber with aluminium foil so that developing solution can generate vapor. TLC silicagel-60plate(Merck)was incubated at 80ºCfor 4 h for activation. After 30 min of TLC chamber preparation, phospholipidsextracted from C. glabratacells werespotted at thebottom (1.5 cm fromthelowerend) of the
    3. Seperation of phospholipids by thin layer chromatography(TLC)
    4. PI-3kinase reaction was set up ina total volume of50μlin a 1.5 ml microcentrifuge tube as described below.PI-3 kinase reaction buffer = 25 μlSpheroplast lysate = 20 μl (equivalent to 10 μg protein)Sonicated phosphatidyl inositol = 5 μlReaction mix was incubated at 25ºC for 20 min and enzyme reaction was stopped by adding 80μlHCL (1N) solution. To extract phospholipids, 160 μl chloroform:methanol (1:1) was added to the reaction mix withcontinuous mixing. Organic phase containing phopholipidswas separated fromaqueous phase by centrifugation at 7,500g for 4 min at 4ºC and transferred to a new vial. Using vacuum evaporator apparatus, solvent was evaporated and phospholipidsweredissolved in 10 μl chloroform
    5. PI-3 kinase reaction set up and phopsholipid extraction
    6. 10 mg phosphatidylinositol-sodium salt(from Glycine max)was dissolved in 2 ml chloroform to prepare a 5 mg/ml stock solution. This solution was prepared in a small glass vial aschloroformis known to reactwith polypropylene. Small aliquots of stock solution were madeand stored at -20ºC till further use. To avoid spillage due to vapor pressure, vials containing phosphatidylinositol-sodium salt solutionwereopened very carefully.To prepare sonicated phosphatidylinositolfor one PI-3 kinase reaction, 2 μlof the stock phosphatidylinositolsolution (10 μg) wastransferredtoanew1.5 ml microcentrifuge tube. Using vacuum evaporator apparatus, chloroformwas evaporated from the solution and phosphatidylinositol-sodium saltwas resuspended in 5 μl sonication buffer.For sonication, a total of 20 pulses, each of 30 sec with30 sec resting time weregiven on ice
    7. Preparation and sonication of phosphatidylinositol-sodium salt solution
    8. A single colonyof desired C. glabratastrainwas inoculated in YPD-liquid mediumand grown for 14-16 h. 50 μl overnight culture was inoculated inYPD-liquid mediumfor 4 h. Log-phase-grownyeast cells were harvested,washedwith PBSandwereinoculated atinitial OD600of 2 and 4,into YNB-dextrose and YNB-sodium acetate liquid medium,respectively.After 4 hincubation,yeast cells were harvested by centrifugation at 2,500g for 5 minand treated with 1.2 M zymolyasefor 1 hto obtain spheroplasts.Post zymolyase treatment, spheroplasts were resuspended in 100 μl resuspension bufferandanequal amount of 0.25 mm glass beadswasadded to lyse the spheroplasts. Using bead beater apparatus, spheroplasts were lysed and protein concentration in spheroplast lysateswas determined usingbicinchoninic acid assay (BCA) method and samples were stored at -20ºC till further use
    9. Preparation of cell lysate
    10. In vitroPI-3 kinase reactions wereset up to measure PI-3P synthesized as described earlier(Whitman et al., 1988)
    11. Phosphatidyl inositol-3 kinase (PI-3 kinase assay)
    12. C. glabrata cells were grown overnight in 10 ml YPD liquid medium. Cells were harvested at 2,500g for 5 min, resuspended in 400 μl Buffer A (50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 1% SDS) and were transferred to a 2 ml microcentrifuge tube. Equal volume of phenol-chloroform-isoamyl alcohol (PCI) solution was added tocell suspension and tubes were vortexed for 2-3 min. After incubation at 42 ̊C for 30 minon a thermomixer set at 800 rpm (Eppendorf), cell debris was removed by centrifugation at 7,500g for 10 minand aqueous phase (300-350 μl) was carefully transferred to a new 2 ml microcentrifuge tube. Genomic DNA was precipitated with800 μl chilled absolute ethanol and 35 μl sodium acetate (3 M, pH 5.2). DNA pellet was washed with chilled 70% ethanol and dried at room temperature for 5-10 min. Genomic DNA pellet was dissolved either in 50 μl 0.1X TE or molecular biology grade water containing 0.3 μl Ambion RNase cocktail and incubated at 37 ̊C for 30 minfor digestionof RNA. After RNA degradation, 100 μl of 0.1X TE or nuclease-free water was added to the tube and stored at -20ºC. Quality of extracted genomic DNA was checkedon 0.6% agarosegel by electrophoresis
    13. Genomic DNA isolationby quick genomic DNA extraction method
    14. C. glabratastrains were grown overnight in YPD medium. Cellswereharvested from 1 mlcultureandwashed withPBS.Cells were next washed with50mM NaH2PO4andresuspendedin 100 μlFITC-dextran(50mg/ml). After incubation at 37°Cfor 45 min, cells were washed thrice with PBS for complete removal of FITC-dextran.Yeast cells were resuspended in 1 ml PBS and used to infect PMA-treated THP-1 cells in 4-chambered glass slide
    15. Fluorescein isothiocyanate(FITC)staining of C. glabratacells
    16. For confocal microscopyanalysis, 5X105THP-1 cells were seeded and treatedwithPMA in 4-chambered slides. Differentiated THP-1 macrophageswere infected either with FITC-labeled or GFP-expressingC.glabratastrains to a MOIof 1:1. At different time intervals, medium was aspirated out from each chamber of 4-chambered slides and chamberes were washed twice with PBS. To fixthe infected macrophages,500 μlformaldehyde(3.7%) was added gently toeach chamber andincubated for 15 minat room temperature. Each chamber of the slide was washed twice withPBS to remove formaldehyde solution completely. To permeabilize the fixed cells, 500 μl Triton-X (0.7%) was dispensed toeach chamber and slide wasincubated at room temperature for 5 min. Chambers of the slide werewashed twice with PBS, separated from the slideusing a chamber removal device andwere air dried. Coverslips were placed onslides using Vectashield mounting mediumand bordersweresealed withnail paint. Slides werestored at 4°C until used forfluorescence imaging
    17. Fixing of PMA-treated THP-1 macrophages
    1. Adherent cells growing either on cover slips or chamber slides were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed with PBS thrice for 5 min each and blocking was done in 2% BSA(preparedin PBScontaining 0.3% Triton-X 100) for 1h.The cells were incubatedwith primary antibody(dilutedin PBScontaining 0.3% Triton-X 100)for 2h at room temperature or overnight at 4⁰C.The cells were washed with PBS thrice for 5 min each followed by incubation withAlexa Fluor 488-or 594-conjugatedsecondary (anti-mouse/rabbit) antibodiesfor 1h. Then the cells were mounted on microscopicslides using Vectashieldmountingmediumcontaining nuclear dye DAPI. Imaging was done byeither the laser scanning confocal LSM510 or LSM 750 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscope
    2. Immunofluorescence Microscopy
    3. Neutralization solution(Solution III)
    4. Lysissolution(Solution II)
    5. Resuspension solution(Solution I)
    6. For Plasmid isolation
    7. Blocking Buffer
    8. NP-40ComponentsFinal concentrationFor 10 mlNP-4010%1mlH2O9ml
    1. Bradford method(Bradford, 1976)was used to determine the quantity of protein in various samplesin a 96-well plate. Bradford’s reagent was prepared by diluting Bradford dye with water in the ratio of 1:5.For estimating the concentration of protein in a particular sample, 50μl volume reactionwas set and200μl of freshly prepared Bradford’s reagent was added. The complex givesa purplish colorwhose intensity is proportional to the amount of protein present in the sample. A standard curve was also generated using increasing concentrations of BSA (50 μg/ml, 100 μg/mland 200μg/ml).Cell lysatesof test samples werediluted to1:50 in the same volume.Each sample (including blank and standards) was taken in duplicates.The concentration of protein was measuredusing the ELISA reader at 570 nm. The unknown protein concentration (X) was calculated as follows:where,OD1& OD2: Optical densities of Standard (Std) 1 & Standard (Std) 2, respectively.BSA: Bovine serum albuminX×50 (dilution factor)/1000 = YConcentration of unknown protein (μg/μl) = Y × OD
    2. Estimationof protein concentration in cellular lysates
    3. 6XEMSA sample loading dye
    4. 5X EMSA buffer
    5. Native EMSA PAGE
    6. 10XBinding buffer
    7. For Electrophoretic Mobility Shift Assay (EMSA)
    8. TBS-T
    9. Transfer buffer
    1. placed on ice.The samples were centrifuged at 10,000x gfor 10 min at 4°C and the supernatant was extracted by avoiding the glass beads. Absorbance of thelysatewas measuredat 260 nm, and it was divided into 200 μL aliquots and frozen at -80°C. No difference in the ribosome profiles were detected between thesesamples and fresh samples analysed immediately.Polysomes were analysed by centrifugation through a 10-50% sucrose continuous gradient. The gradient was prepared by pipetting, gradient buffers (Section 2.1.6.3), 5 mLof each layer onto the bottom of an 11 mLopen top centrifuge tube, covering the tube with paraffin filmand by placing it horizontally on a flat surface at 40C for 2 h. The ribosome sample (cell lysate) was loaded on top, and gradient was centrifuged at 100,000x gat4°C for 6 h in an SW41 rotor (Beckman). Anamount of lysate equivalent to 10 A260units was loaded on the gradient. Ribosome levels were measured by the gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Polysomepurification was performed by layering the cell lysate on 37% sucrose solution in an 11 mLopen top centrifuge tube, and centrifuged at 100,000x gfor 14 h in an SW41 rotor(Beckman). The pellet was suspended in 50 to 100 μL of lysis buffer. An amount oflysate equivalent to 0.7A260unitswas loaded on the gradient. Ribosome levels were measured by gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Ribosome subunits were analysedby centrifugation through a 10-30% sucrose continuous gradient. The gradient was prepared by pipetting 2.2 mLof each layer onto the bottom of a 5 mLopen top centrifuge tube, sealing it with paraffin filmand placing it horizontally on a flat surface at 4°C for 2 h.The ribosome sample wasloaded on top, and gradient was centrifuged at 100,000x gand 40C for 4.5 hin an SW55rotor (Beckman). Ribosome subunit levels were measured by gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Ribosome profiles with yeast cell lysates and purification of ribosomes from yeast were performed with the help of Mr. Aluri Srinivasand Dr. Umesh Varshney
    2. The method to analyse ribosome profiles was adapted from (Leeet al., 1992). Yeast cells were grown to 0.2 to 0.8 OD600at 30°C in 200mLof YPD,and cycloheximide was added to this media at 50μg/mLfinal concentration. The culture was placedand mixed continuously onanice and salt mixture for 2-5 min and centrifuged immediately at 4,000 x g. The culture was not allowed to stay for a longer time on the ice and salt mixture to avoid freezing. The cell pellet wassuspended in 1mLof lysisbuffer (Section 2.1.6.3),andtransferred to a2 mL microfuge tube, to which1mLglass beads (0.45-0.6mm diameter)were added, and lysed by bead beating for 10 min with intervals (30 sec on time and 1min off time).During the 1 min off time, tubes were
    3. Ribosome profiles
    4. 20 mM HEPES500 mM NaCl 2 mM EDTA1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer C)IP7 reaction buffer(10X)250 mM HEPES,pH 7.4500 mM NaCl60 mM MgCl210 mM DTT (1 M stock was made separately, aliquoted into 100 μL and stored at -20oC).10X buffer was made and stored at 4oC. An appropriate amount was added to the reaction mix to get a final concentration of 1X.DTT was added fresh to the reaction buffer just before use
    5. Buffer C
    6. 2 mM EDTA5 mM DTT1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer B)
    7. 20 mM HEPES pH 6.8100 mM NaCl
    8. Buffer B
    9. 20 mM HEPES pH 6.8100 mM NaCl2 mM EDTA5 mM DTTYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer A)
    10. Buffer A
    11. Buffers for protein purification and IP7reaction
    1. lysed with ice-cold NETN lysis buffer (20mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5% Nonidet P-40) containing protease inhibitors (0.5 mM PMSF, 1 mg/ml aprotinin and 1 mg/ml pepstatin) for 30 min.on ice, and then centrifuged at 14000 RPM for 10 min. at 4°C. Protein concentration in the supernatant (cell lysate) was estimated by Bradford assay. 20ug of the protein lysate was mixed with6X SDS loading dye (100 mMTris pH6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol and 200 mMβ-mercaptoethanol), boiled for 5 minutes at 99°C. Protein samples were resolved electrophoreticallyon SDS-polyacrylamide gels and transferred onto PVDF-membrane (Amersham Biosciences) using a semi-dry transfer apparatus (Biorad) for 1h at a constant current of 500 mA. Membranes were blocked for 30 min.with 5% non-fat dry milk powderdissolvedin 1X PBS and then immunoblotted overnight with primary antibody diluted in blocking buffer. Membranes were washed four times (each 5 min.) with 1X TBST followed by incubation with secondary antibody conjugated with horseradish peroxidase (HRP) for one hour at room temperature. The membrane was washed four times with 1X TBST (each wash for 5 min.), and the specific proteins on the membrane were detected by using enhanced chemiluminescent (ECL) reagents in 1:1 ratio (Amersham)