CHAPTER 4 RESULTS PAGE

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					                                       Chapter 4


CHAPTER 4                RESULTS                                         PAGE

   4.1 CHARACTERIZATION OF THE LPS                                        80
     4.1.1. KDO determination                                             80
     4.1.2. Determination of the carbohydrate content                     81
     4.1.3. Determination of the protein content                          82
     4.1.4. Electrophoretic analysis of LPS                               83
   4.2 AEQUORIN-LUMINESCENCE MEASUREMENTS OF                              84
                               2+
      INTRACELLULAR Ca
     4.2.1. Intracellular Ca2+ influx by the Ca2+-ionophore A23187        84
     4.2.2. YE-induced Ca2+ influx                                        86
     4.2.3. LPS-induced Ca2+ influx                                       87
   4.3. MEASUREMENT OF THE OXIDATIVE BURST REACTION                       89
     4.3.1. The luminol-chemiluminescence assay                           89
       4.3.1.1. The YE- and LPS-induced oxidative bursts                  90
       4.3.1.2. Inhibition of the oxidative burst reaction by catalase    91
       4.3.1.3. The effect of LPS pre -treatment on the YE-induced        92
              oxidative burst reaction
     4.3.2. The H 2DCF-DA fluorescence assay                              93
       4.3.2.1. Kinetics of the H2DCF-DA-measured oxidative burst         94
       4.3.2.2. YE and LPS concentration studies                          95
       4.3.2.3. Inhibition of the YE- and LPS-induced oxidative bursts    98
        4.3.2.3.1. N -acetyl-L-cysteine (NAC)                             98
        4.3.2.3.2. Diphenylene iodonium (DPI)                             100
        4.3.2.3.3. Diethyldithiocarbamate (DDC)                           101
        4.3.2.3.4. 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-      103
                  oxide (PTIO)
       4.3.2.4. Effect of protein kinase - and protein phosphatase        104
              inhibitors on the oxidative burst
   4.4. THE ROLE OF Ca2+ IN THE ELICITOR -INDUCED OXIDATIVE               108
       BURST




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     4.5. EXTRACELLULAR ALKALINIZATION                                                         110
        4.5.1. YE and LPS                                                                      110
        4.5.2. Effect of protein kinase- and protein phosphatase inhibitors                    111
              on the extracellular alkalinization
     4.6. INDUCTION OF A REFRACTORY STATE IN TOBACCO                                           113
          CELLS
     4.7. PROTEIN PHOSPHORYLATION STUDIES                                                      115
              32
        4.7.1. [ P] orthophosphate uptake study                                                115
        4.7.2. Phosphoprotein induction by YE and LPS                                          116
        4.7.3. Tricine-SDS-PAGE of LPS-induced phosphoproteins                                 119
        4.7.4. The effect of protein kinase- and protein phosphatase                           120
              inhibitors on protein phosphorylation


                              LIST OF FIGURES AND TABLES

FIGURE / TABLE                                                                               PAGE

FIGURE 4.1:    Standard curve and absorbance spectrum for KDO determination                    80


FIGURE 4.2:    Standard curve and absorbance spectrum for carbohydrate determination           81


FIGURE 4.3:    BSA standard curve for protein quantification                                   82


FIGURE 4.4:    SDS-PAGE of Burkholderia cepacia and Ralstonia solanacearum LPS                 84


FIGURE 4.5:    Cell permeabilization experiment of aequorin-transformed tobacco cells          85


FIGURE 4.6:    Effect of YE on intracellular Ca2+ influx                                       87


FIGURE 4.7:    Effect of LPS on intracellular Ca2+ influx                                      88


FIGURE 4.8:    Effect of Ficoll 400 on chemiluminescence of the YE-induced oxidative burst     90



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                                                     Chapter 4

FIGURE 4.9:       Luminol-chemiluminescence of the YE- and LPS-induced oxidative bursts       91


FIGURE 4.10:      Inhibition of the YE-and LPs-induced oxidative bursts by catalase           92


FIGURE 4.11:       Effect of LPS pre-treatment on the YE-induced oxidative burst              93


FIGURE 4.12:      Kinetics of the H2DCF-DA fluorescence assay                                 94


FIGURE 4.13 (A): Time course of the YE-concentration-dependent ROS production                 96


FIGURE 4.13 (B): Maximum ROS production during the YE-concentration study                     97


FIGURE 4.14:       Maximum ROS production during the LPS concentration study measured         98
                  by the H2DCF-DA fluorescence assay


FIGURE 4.15:      NAC concentration-dependent inhibition of the YE-induced oxidative burst    99


FIGURE 4.16:      DPI concentration-dependent inhibition of the YE-induced oxidative burst    100


FIGURE 4.17:       DDC concentration-dependent inhibition of the YE-induced oxidative burst   101


FIGURE 4.18:      Inhibition of the LPS-induced oxidative burst by NAC, DPI and DDC           102


FIGURE 4.19:       Effect of the nitric oxide scavenger, PTIO, on the YE- and LPS-induced     104
                  oxidative bursts


FIGURE 4.20:       Effects of protein kinase - and protein phosphatase inhibitors on          106
                  the YE-induced oxidative burst


FIGURE 4.21:       Effects of protein kinase - and protein phosphataseinhibitors on           107
                  the LPS-induced oxidative burst




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FIGURE 4.22:   The role of Ca2+ during the YE induced (A) and LPS-induced (B) oxidative     109
               bursts


FIGURE 4.23:   YE- and LPS-induced extracellular alkalinization of tobacco culture medium   111


FIGURE 4.24:   The effect of protein kinase - and protein phosphatase inhibitors on         112
               the YE-induced extracellular alkalinization


FIGURE 4.25:   The effect of protein kinase - and protein phosphatase inhibitors on         113
               the LPS-induced extracellular alkalinization


FIGURE 4.26:   The induction of a refractory state in tobacco cell cultures by YE and       114
               LPS pre-treatment


FIGURE 4.27:   Time-dependent [ 32P] orthophosphate uptake by N. tabacum cell cultures      116


FIGURE 4.28:   SDS-PAGE (15%) and autoradiogram of phosphoproteins induced by YE            117


FIGURE 4.29:   SDS-PAGE (15%) and autoradiogram of phosphoproteins induced by LPS           118


FIGURE 4.30:   Tricine SDS-PAGE (15%) and autoradiogram of phosphoproteins                  120
               induced by LPS


FIGURE 4.31:   SDS-PAGE and autoradiogram of the effect of protein kinase - and protein     121
               phosphatase inhibitors on the phosphoproteins induced by YE and LPS


TABLE 4.1:     Characterization of the LPS isolated from Burkholderia cepacia               83


TABLE 4.2:     Summary of the results for intracellular Ca2+ determination                  89


TABLE 4.3:     Summary of the inhibition of the YE- and LPS-induced oxidative bursts        107




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