The reaction was carried out by incubating at 37⁰C for 30 min. The reaction was stopped by adding 2μl of 0.5M EDTA, pH 8.0 and keeping on ice. A spin column was prepared using 1ml syringe and packed with sterile Sephadex G50 slurry and reaction mixture is applied on the top. The eluate is collected in different microcentrifuge tubes and radioactivity was counted using Geiger counter. The tube showing 7 to 9X106was used for experiment. The column containing the unincorporated [γ-32P] ATP was discarded in radioactive waste bin. The radiolabelled oligonucleotides were annealed with their corresponding complementary unlabelled oligonucleotides. A 50 fold molar excess of the latter was used for annealing for conversion of labelled single strand to double strand. Thetubes were kept in boiling waterbath for 3 min followed by room temperature for 30 min. The tubes were transferred to ice and the oligonucleotides were diluted to 4fmoles/μl using sterile H2O

The oligonucleotides were labelled at their 5'end with 32P using T4 polynucleotide kinase (T4 PNK) enzyme in a reaction given belo

end labelling of the oligonucleotides

and stained with ‘Live & Dead’ cell assay reagent (5 μM ethidium homodimer, 5 μM calcein-AM) for 30 min at room temperature. Red (as dead) and green (as live) cells were analyzed under a fluorescence microscope (Labophot-2, Nikon, Tokyo, Japan).For flow cytometry, cells were transiently transfected with either empty vector or various constructs. After 12 h, cells were treated with a combination of CHX (cycloheximide, 25 μg/ml) and TNF (5 nM) for 24 h. Cells were washed, trypsinised and then subjectedto flow cytometry (FACS Aria, BD Biosciences) using Live-Dead Cytotoxicity assay kit (Invitrogen). Live versus dead cells were analysed using FlowJo software

Cytotoxicity assay: The drug-induced cytotoxicity was measured by the 3-(4,5-Dimethylthiazolyl-2)-2,3-diphenyltetrazoliumbromide (MTT) assay as essentially described by Mosmann et al., 1983. Briefly, 5x104cells/well were seeded in 96-well plate. After 12 h, cells were treated in triplicates with different agents for different concentrations and time (in a final volume of 100μl). After completion of treatment, 25 μl of MTT solution (5 mg/ml in PBS) was then added and incubated for 2 h. The cytotoxicity was evaluated by uptake and cleavage of yellow MTT dye to purple formazan crystals by dehydrogenase activity in mitochondria of the living cells. Thereafter, 100 μl of extraction buffer (20% SDS in 50% dimethlylformamide) was added. After an overnight incubation at 37ºC, the absorbance at 570 nm was measured using 96-well multiscanner autoreader(Bio-Rad) with the extraction buffer as blank. Absorbance values were normalized to untreated cells and represented in percent cell viability for different concentrations or treatments.Determination of nuclear fragmentation: The morphology of live and dead cells was observed by staining the nucleus with DNA intercalating dye, propidium iodide (PI).Briefly, cells were treated with several apoptotic inducers for different concentration or time. Thereafter, cells werewashed with PBSand fixed in ice-cold 80% methanol for overnight at 4°C. Following day,cells were washed and suspended in 100 μl of PI solution (0.1% Triton, 0.2 mg/ml RNase A and 50 μg/ml PI in PBS) for 30 min in dark. Cells were then mounted on slides and viewed under fluorescence microscope (in 560 nm filter) to determine morphology of intact or fragmentednucleus.Live &dead assay: The cytotoxicity of various drugs was determined using the commercially available Live/Dead assay kit (Molecular Probes, Eugene, OR). Live cells have intact membraneand active cellular metabolism,which allow Calcien-AM to permeate inside and get cleaved into green fluorescent compound, Calcein (ex/em ~495 nm/~515 nm) due to intrinsic cellular esterase activity. On the other hand, Ethidium homodimer-1 (EthD-1) enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em ~495 nm/~635 nm). Hence, the cell viability can be assayed by either flow cytometry or fluorescence microscopy.For imaging, cells with different drugs treatments were washed with PBS

Assays for Apoptosis

Yeast were grown in appropriate medium while the logarithmicphase and 1 OD600cells wereharvested and chilled on an ice(Elion and Warner, 1986). Allsubsequent steps,unless specified, werecarried out at4°C or on ice. Cells were collected by centrifugation at 2500 gfor 6min and washed with2 mLof TMN (Section 2.1.6.4). Cells were suspended in 1 mL of ice cold permeabilization buffer (Section 2.1.6.4) and incubated for 15 min.Cells were pelleted and incubated with 100μLof transcription assay buffer (Section 2.1.6.4) containingradiolabelled [α-32P]UTP. After incubation for 10 min at30°C and 300 rpm in a shaking drybath (Eppendorf),1mLof cold TMN containing1 mM nonradioactive UTP was added, the cells were collected by centrifugation, and RNA was prepared by the hot phenol methodas described in Section 2.2.8. Equal counts of labelled RNA were used for hybridization. The membrane was pre incubated with 50 mL of hybridization buffer for 20-30 min, and hybridization was performed with labelled RNA at 65°C for 15 h in 20 mL of hybridization buffer

Transcription run on analysis

immunoprecipitation by using substrate specific antibody or pull-down by affinity trapping the substrate tag. The IP/pull-down complexes wereanalyzed by detecting the ubiquitination of substrate protein by using either substrate specificantibody or ubiquitin antibody through western blotting

HeLa cells were transfected with various combinations of plasmids. At 24 h post-transfection, cells were treated with MG132 (10μM) for 6 h and the whole-cell extracts were prepared by NETN lysis. The cell lysatewas subjected to

In vivoubiquitylation assay

2 bed volumes of methanol, and equilibrated with 5 bed volumes of distilled water. In order to reduce the water solubility of siderophore in the supernatant, it was acidified to pH 2 using concentarted HCl. This acidified supernatant was passsed through the column, and finally eluted with 160 ml methanol by collecting approximately 60 fractions (2 ml each) of the flow through. Siderophore assay was done on CAS plate with each collected fraction. Fraction that gave orangish-yellow halo for the siderophore on CAS plate, was combined together, dried in rotary evaporator and finally reconstituted in 1 ml methanol for further quantification using HPLC as described previously (Amin et al., 2009).For HPLC analysis, siderophore samples were filtered through filter membrane (porosity, 0.45 μ). Next, 10 μl sample was injected into Agilent C18 (4.6mm×250mm×5μm) column (gradient:(A=H2O/0.1%TFA), (B= CH3CN/0.1%TFA) 0-30% B in 10 min, 30-45% B in 15 min,45-0%B in 20 min at a flow rate of 1 ml/min). Similarly, standard vibrioferrin (siderophore produced by Xanthomonas) was also estimated through HPLC for comparison. Fe(III) bound vibrioferrin complex was prepared by incubating FeCl3.6H2O and apo-vibrioferrin for overnight. This complex was detected at300 nm (RT 10.998 min), whereas apo-vibrioferrin was detected at 220 nm at RT 10.988 min. The siderophore concentration in the samples were determined by peak area and calculated against the standard curves obtained from standard vibrioferrin. The siderophore from the test samples were detected at 300 nm, which confirms that majority of the vibrioferrin isolated from the culture was present in bound form

Different Xanthomonas oryzaepv. oryzicola strains were grown overnight in PS medium at 28 °C and 200 rpm. 0.2% of the overnight grown culture was inoculated in the the fresh PS medium supplemented with 50 μM 2, 2’-dipyridyl, and grown till OD600 reached to 1. Cultures were centrifuged at 12,000 g for 50 min to get the cell free culture supernantant, which was collected into acid treated bottles. Excess exoplysaccharide was removed by centrifugation for longer time. Siderophore was initially isolated by column chromatography as described previously (Wright, 2010). Briefly, 220 g of XAD-16 resin was soaked overnight and packed into the column (2.4×30 cm), column was wa

HPLC based siderophore estimation from culture supernatant of different strains of X. oryzaepv. oryzicola

Cells were seeded in six well plates. After attaining optimal growth, cell lysates were prepared by scraping cells in 1x Laemmli buffer and samples were processed by standard western blot techniques. To detect FAKactivation samples were processed as described in Section 2.2.13. Briefly, 2 x106cells (6 x105cells per time point) were held in suspensionin complete medium containing 1% methylcellulosefor 90 min (Susp), andreplatedon fibronectin (2 μg/mL) coated surfacesfor 20 min (+FN)or for 4 h (SA -stably adherent). Cells at each time point were lysed in 1x Laemmli buffer and subjected to immunoblotting. Membranes were probed with specific antibodies(Table 2.3)and detected using the ECL detection system (GE Healthcare) as described in Section 2.2.10

Immunoblot analysis to detect pFAK

April 15, the day when you pay your taxes, gives you a good index of how democracy is functioning. If democracy were functioning effectively, April 15 would be a day of celebration. That’s a day on which we get together to contribute to implementing the policies that we’ve decided on. That’s what April 15 ought to be. Here it’s a day of mourning. This alien force is coming to steal your hard-earned money from you. That indicates an extreme contempt for democracy. And it’s natural that a business-run society and doctrinal system should try to inculcate that belief.

ZFIN_ZDB-ALT-110210-15

prayed unto the Lord, yea, even with all his heart

DISOLUCIÓN ANTICIPADADE LAS CÁMARAS

We should, probably, in time aspire to have foreign relations of our own, to have our own army and navy, and to seek for that complete emancipation which with communities as with individuals, maturity prompts. But independence in a state must always be relative, and none of us can expect to live to see the day when the British dominions in this part of the world will be peopled to such an extent, and become so powerful, that they can afford to be independent of England. We must, from the necessities of our geographical position—so long as the United States continue to be as powerful as they are ; and even if they were divided into two or three portions—we must always find in them a source of danger which must force upon us a dependence on England.

Speaking of the Conference at Quebec, he stated that “the delegates unanimously resolved that the United Provinces of British North America shall be placed at the earliest moment in a thorough state of defence.” Hon. gentlemen, I was not aware that the Imperial Government had ever cast off the burden of the defence of this province.

when the time comes in the history of any colony that it has overcome the burdens and embarrassments of early settlement, and has entered on a career of permanent progress and prosperity, it is only fair and right that it should contribute its quota to the defence of the Empire.

11

CACNA1C

The incorporation of private or local companies, except such as related to matters assigned to the General Parliament, would be reserved to the local Governments, being matters of a local character. Even the present law permitted the incorporation of companies under a very simple system, which would probably be continued.

It was desirable the General Government should have the control of the medium through which the trade and commerce of the country was carried on, and that in the establishment of banks, the issue of paper money and in offering to the public the paper representative of their labor, in whatever part of the country, there should be the same legislative security for the people

The control of the Militia was certainly a subject which they must all feel ought to be in the hands of one central power. If them was one thing more than another which required to be directed by one mind, governed by one influence and one policy, it was that which concerned the defence of the country.