Calcium Transport

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Click Here For Research Papers Online! PHM499 Research Project Calcium transport study of SF-9 lepidopteran cells and bull frog sympathetic ganglion cells ABSTRACT The intracellular calcium level and the calcium efflux of the bull-frog sympathetic ganglion cells (BSG) and the SF-9 lepidopteran ovarian cells were investigated using a calcium-sensitive fluorescence probe fura-2. It was found that the int INTRODUCTION Spodoptera frugiperda clone 9 (SF-9) cells are a cultured insect cell line derived from the butterfly ovarian tissue. SF-9 cells are used by molecular biologists for the studies of gene expression and protein processing (Luckow and Summers, 1988). Howe It was found that the SF-9 cells appeared to have a calcium concentration similar to the BSG cells. Moreover, the calcium extrusion rates of both cell types with no Na2VO4 added seemed to the same. However, due to insufficient data, the effects of Na2V After obtaining these basic parameters, many questions raised such as how does the SF-9 cells extrude their calcium and why the Na2VO4 affected the calcium efflux for the SF-9 cells but not the BSG cells? The SF-9 cells may have a calcium pump or excha MATERIALS AND METHODS Chemicals and solutions 4-bromo-A23187 and Fura-2/AM were purchased from Molecular Probes (Eugene, OR). Na2VO4 was purchased from Alomone Lab (Jerusalem, Israel). Dimethyl sulfoxide (DMSO) was obtained from J. T. Baker Inc. (Phillipsburg, NJ). All other reagents were obtaine The normal Ringer's solution (NRS) contained (mM): 125 NaCl, 5.0 KCl, 2.0 CaCl2, 1.0 MgSO4, 10.0 glucose, 10.0 N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES). The calcium free Ringer solution (0CaNRS) is the same as the NRS except CaC Fura-2/AM solution was prepared as follows: a stock solution of 1mM fura-2/AM in DMSO was diluted 1:500 in NRS containing 2% bovine albumin. It was then sonicated for 10 minutes. It was then kept frozen until the day of the experiment. 20 SYMBOL 109 \f "Symbol"M 4-bromo-A23187 solution was prepared by diluting a stock of 5mM 4-bromo-A23187 in DMSO 1:250 with NRS. Na2VO4 solution (VO4NRS) contained 100 SYMBOL 109 \f "Symbol"M. Na2VO4 in 0CaNRS. All experiments were performed at room temperature, 22-26 SYMBOL 176 \f "Symbol"C. The above solutions were adjusted to pH 7.3 with NaOH. Cells BSG cells were obtained as described by Kuffler and Sejnowski (1983). BSG cells were plated and incubated at 3-10 SYMBOL 176 \f "Symbol"C for up to 4 days before the experiments. The cells were plated on custom made 3.5 cm plastic culture dishes. A SF-9 cells (non-transfected) were cultured as described by Summers and Smith (1987). The SF-9 cells were plated and incubated (at 37 SYMBOL 176 \f "Symbol"C) on the custom made dishes as used for the BSG cells one day prior to the experiments. They w Each dish contained approximately 100 SYMBOL 109 \f "Symbol"l of cell suspension. To load the cells with fura-2/AM, 100 SYMBOL 109 \f "Symbol"l of fura-2/AM /BSA solution was added for 30 minutes. Intracellular calcium measurements Fura-2 is a fluorescence indicator of calcium that is used to determine the free intracellular calcium concentration. Fura-2/AM was used in the experiments instead of fura-2. Fura-2/AM is an ester moiety of fura-2 which has the advantages of being perm The apparatus included a fluorescence microscope unit and a spectrofluorometer system. The fluorescence microscope unit consisted of a 75 W Xenon arc lamp and a Zeiss inverted microscope with a Zeiss Neofluor 63X objective. In addition, a pipette was placed close to the sample cells (within 5mm) for perfusion. The pipette delivered the s The PTI Deltascan 4000 microscope system (Photon Technology International Inc., South Brunswick, NJ) was used to make fluorescence measurements. Emitted fluorescence signal was detected by the photomultiplier tube (PMT) and recorded via a NEC 286 microc The experimental methods of calcium measurements used in the experiments were similar to the one described by Schwartz et al. (1991). In brief, intracellular free calcium concentration can be determined through the following formula (Grynkiewicz et al. [Ca2+]i = Kd.(Fmin/Fmax).(R-Rmin)/(Rmax-R) where Kd is the effective dissociation constant for the Ca2+-fura-2 complex, Fmin and Fmax are the fluorescence intensities at SYMBOL 108 \f "Symbol"=380nm obtained from calcium-free fura-2 sample and calcium-bound fura-2 sample respectively, R is the f One average size cell from each dish was randomly selected for the measurement. NRS was initially perfused to wash out the fura-2/AM in the cell suspension. When the intracellular calcium level was stabilized, it was switched to 2-bromo-A23187 to raise Statistical Analysis Statistical analysis was performed with using The Student Edition of Minitab release 8 (Minitab Inc., 1991). Results It was found that the intracellular calcium concentration in the SF-9 cells was 44.7 ± 8.3 nM (mean ± S.E., n = 8) in NRS. The calcium concentration in the BSG cells was found to be 58.2 ± 9.0 nM (n = 4). Student's t test did not indicate a significant The rates of active transport of calcium out of the cells following 0CaNRS were also calculated. They were determined by performing a linear regression on the linear portion (ranging from 20 - 50 seconds) of the decline following the maximum calcium con However, the BSG cells and the SF-9 cells were generally in different sizes in which the SF-9 cells (about 15-20 SYMBOL 109 \f "Symbol"m in diameter) were usually smaller in sizes relative to the BSG cells (about 25-40 SYMBOL 109 \f "Symbol"m in diam J = -SYMBOL 68 \f "GreekMathSymbols"C/SYMBOL 68 \f "GreekMathSymbols"t ·V/S where J is the flux, -SYMBOL 68 \f "GreekMathSymbols"C/SYMBOL 68 \f "GreekMathSymbols"t is the rate of calcium depletion and V/S is the volume to surface area of the cell (V/S can be further simplified to r/3 where r is the radius of the cell). The calculated calcium efflux of the BSG cells and the SF9 cells were 2.02 ± 0.44 fmole·cm-2·s-1 (n = 10) and 1.33 ± 0.26 fmole·cm-2·s-1 (n = 7) respectively (table 1). There was no significant difference between the two efflux values (P = 0.2) shown b Similarly, the rates of calcium depletion of the BSG cells and the SF-9 cells following VO4NRS were 9.24 ± 0.22 nM/s (n=2) and 2.46 ± 0.75 nM/s (n=3) respectively. The adjusted calcium efflux of the BSG cells and the SF-9 cells were 6.00 ± 0.14 fmole·cm In addition, it was observed that SF-9 cells lost the ability to extrude the calcium after two to three cycles of VO4NRS applications (Figure 1). On the other hand, the BSG cells did not appear to lose their abilities to extrude the calcium after up to Table 1 Rate of Calcium depletion of BSG and SF-9 cells after the addition of 0CaNRS BSG rate of calcium depletion (nMs-1) BSG calcium efflux (fmole·cm-2·s-1) SF-9 rate of calcium depletion (nMs-1) SF-9 calcium efflux (fmole·cm-2·s-1)  2.23 1.01 4.67 1.51  0.54 0.24 4.10 1.33  4.36 1.98 3.19 1.03  8.58 3.89 7.74 2.51  5.88 2.67 5.55 1.80  1.28 5.81 2.01 0.65  5.28 2.40 1.56 0.50  7.02 4.55    2.22 1.44    2.27 1.47     Intracellular calcium concentration of a single sample cell was raised using 4-bromo-A23187 and was subsequently lowered by introducing 0CaNRS. These data represented the rates of decline (SYMBOL 68 \f "GreekMathSymbols"C/SYMBOL 68 \f "GreekMathSymbol Table 2 Rate of Calcium depletion of BSG and SF-9 cells after the addition of VO4NRS BSG rate of calcium depletion (nMs-1) BSG calcium efflux (fmole·cm-2·s-1) SF-9 rate of calcium depletion (nMs-1) SF-9 calcium efflux (fmole·cm-2·s-1)  9.02 5.85 1.05 0.34  9.47 6.14 3.59 1.16    2.74 0.89   Similar to Table 1 except VO4NRS was used instead of 0CaNRS to lower the calcium concentration.  Figure 1. Intracellular calcium concentration of a SF-9 cell A time course calcium recording of a single SF-9 cell (19 SYMBOL 109 \f "GreekMathSymbols"m) with the successive applications of 4-bromo-A23187, NRS, 0CaNRS and VO4NRS. It was noted that after 2 applications of VO4NRS, the cell was impaired in its abil Abbreviations: A, 4-bromo-A23187; N, NRS; 0, 0CaNRS; V, VO4NRS.  Figure 2. Intracellular calcium concentration of a BSG cell In contrast to the SF-9 cell in Figure 1, the BSG cell (39 SYMBOL 109 \f "GreekMathSymbols"m) still maintained its ability to extrude (or decrease) calcium after three applications of VO4NRS even at a high calcium concentration. Abbreviations: same as in Figure 1. DISCUSSION In the beginning of the experiment, both the transfected and non-transfected SF-9 cells were used although only non-transfected SF-9 cells were reported here. It was found that the transfected cells had unusual low calcium conc

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