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II-1. Samplllle preparation
A wild-type (WT) strain of Schizosaccharomyces pombe and its mutant that cannot produce CoQ10 due to disruption in the p-hydroxybenzoate polyprenyl diphosphate transefrase gene [11], (denoted ∆ppt1) were kindly provided by Profs. M. Kawamukai and T.
Yamamoto of Shimane University. The WT and ∆ppt1 S. pombe strains were pre-cultured at 30 ºC on a YES plate containing yeast extract (5 g/L), glucose (30 g/L), agar (17 g/L), and amino acids including adenine, histidine, leucine, uracil, and lysine (50 mg/L each). After 3–5 days of cultivation, a large number of colonies of the WT and ∆ppt1 strains were found spreading on the YES plate (Figure II-1). A single colony of the WT and ∆ppt1 strains was harvested from the YES plate and transferred into Pombe Mineral Leucine Uracil (PMLU) medium, which is a minimum nutrition medium with leucine and uracil (75 mg/L each). The PMLU medium was supplied with five different antioxidants—glutathione (abbreviated as GSH), (R)-(+)-lipoic acid (RLA), ascorbic acid (Vit C), and an inclusion complex of (R)-(+)-lipoic acid in γ-cyclodextrin (RLA/γCD)—at 0.5 mM. All the antioxidants were completely dissolved in the medium. The instant when the single colony was added into the fresh PMLU medium with an antioxidant was defined as time zero and Raman spectroscopic measurement commenced. A 200 µL of the prepared sample was then transferred onto a poly-D-lysine coated glass-bottomed dish (MatTek; P35GC-1.5-10-C).
RLA and RLA/γ-cyclodextrin were provided by CycloChemBio. GSH and Vit C were commercially obtained from Sigma. All the chemicals were used as received. The chemical structures of the antioxidants (RLA, GSH, and Vit C) and γ-CD are shown in Figure II-2.
RLA (Figure II-2a) can be reduced to dihydrolipoic acid, thereby showing the antioxidant effects [23]. GSH (Figure II-2b) is a tripeptide containing a thiol group as a reducing agent. In acting as an antioxidant, GSH is converted to its oxidized form, glutathione disulfide (GSSG) [24]. L-Ascorbic acid (Figure II-2c), also known as vitamin C, is a common food additive that is believed to have the antioxidant activity [25]. GSH and Vit C are water soluble, whereas
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RLA is much less soluble in water. To improve the aqueous solubility of RLA, an inclusion complex of RLA with γ-CD was also used in this study. As shown in Figure II-2d, γ-CD is a cyclic oligosaccharide that consists of 8 glucose monomers via α-1,4 linkages. It forms a toroid shape with an inner diameter of ≈9 nm in which a guest molecule can be accommodated.
II-2. Laboratory-built confocal Raman microspectrometer
Figure II-3 is a schematic illustration of the laboratory-built confocal Raman microspectrometer [14, 15] used in this study. The 632.8 nm output of a He-Ne laser (Thorlabs; HRR170) was used as the Raman excitation source. The laser beam was magnified by a factor of ~2.67 in order to effectively cover the exit pupil of the objective used and to better use a high NA of the objective. The expanded laser beam was introduced to a custom-made inverted microscope (Nikon; TE2000-U) by an edge filter (Semrock;
LP02-633RU-25) and a hot mirror (Thorlabs; FM02). The laser beam was focused on the sample cell by an oil-immersion objective (Nikon; CFI Plan Fluor; 100× oil, NA=1.3) placed on the microscope stage, and backward scattered light was collected by the same objective.
The backward scattered light was guided along the opposite direction to the incoming path.
The Rayleigh scattering and anti-Stokes Raman scattering were rejected by the edge filter and only Stokes Raman scattering was transmitted. Then the Stokes Raman scattered light was focused on a 100 µm pinhole by a 150-mm lens and then collimated by another 150-mm lens.
With the use of the 100 µm pinhole and two 150 mm lenses for a confocal configuration, spatial resolution of 308 (±7) nm in lateral (XY) direction and 3.23 (±0.09) µm in axial (Z) direction was achieved. The Stoke Raman scattered light was introduced to a spectrometer (HORIBA Scientific; iHR320) and detected by a liquid nitrogen-cooled charge-coupled device (CCD) detector (Princeton Instruments; Spec-10:100) with 100 × 1340 pixels operating at −120 ºC. A 600 grooves/mm grating was used. The resulting spectral resolution
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was ≈7 cm−1, which was high enough in this work because the Raman spectra of biological samples usually exhibit relatively broad vibrational bands. This grating can cover a wide spectral range over the fingerprint region (>2000 cm-1). For bright-field observation, the sample was illuminated by a halogen lamp (or a mercury lamp) and optical micrographs were acquired by a digital camera (Nikon; DS-Ril) mounted on the microscope.
The laboratory-built confocal Raman microspectrometer can also be equipped with a 3-axis piezoelectric stage (PI; P-563.3CD) or a stage-top incubator (Figure II-4) [16] (Tokai Hit; INU-ONICS-F1) on the microscope stage. The 3-axis piezoelectric stage can translate the sample both horizontally and vertically and was used to determine the spatial resolution of our confocal Raman microscope (see the next paragraph). The piezoelectric stage was controlled by a computer program based on LabVIEW (National Instruments). The stage-top incubator was used to keep the experimental environment including temperature and air flow more stable. It can control the temperature by four independent heaters—stage, top, bath, and lens heaters—and the air flow by a gas-flow device (dry air). The set temperatures of the stage, top, bath, and lens heaters were, respectively, 30, 31.5, 30, and 35 ºC, making the sample temperature kept ≈30 ºC. This incubator can prevent the sample from drying and maintain the same culture conditions, allowing us to perform long experiments.
Figures II-5 and II-6 show the calibration results of the lateral (XY) and axial (Z) resolutions of our confocal Raman microspectrometer. To estimate the lateral resolution, the laser spot was scanned horizontally across a sharp edge of a silicon wafer using the intensity of the 520 cm−1 phonon band of silicon. Similarly, to estimate the axial resolution, the intensity rise of the 1019 cm−1 band of indene at an indene–glass interface was measured. By scanning the laser spot continuously to cross the edge or the interface assumed to be infinitely sharp, a plot of the intensity of the band (either 520 or 1019 cm−1) versus the scanned distance was obtained and it was fitted to a model function f(x). f(x) is a Heaviside step function convoluted with a Gaussian function, which is given by Eq. II-1[17].
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where erf denotes the error function, N is a normalization constant, a is the onset of the step function, σ is the width of Gaussian function, C is a constant. The full width at half maximum (FWHM) is equal to (Eq. II-2): lateral direction and 3.23 ± 0.09 µm in the axial direction. Here the error bars represent fitting precision.
II-3. Experimental conditions in Raman measurements
Yeast cells were cultured in PMLU medium in a shaking incubator at 30 ºC. Growth curves were measured by using a cell counting chamber [16] (Marienfeld; 06 401 30). The yeast cells were immobilized well on the poly-D-lysine coated glass-bottomed dish. The excitation laser power was 3.7 mW. The exposure time for each spectrum was 5 s. About 60–70 Raman spectra could be measured in 1 h and the sample dish was replaced every 1 h by a new one that was prepared in exactly the same manner. At each measurement time, a total of 200 Raman spectra were acquired from 200 different S. pombe cells.
II-4. Singular value decomposition analysis
In our experiment, we measured a large number of Raman spectra (200 spectra) so we needed to shorten the exposure time (5 s for each spectrum). To improve the signal-to-noise ratio in the spectra acquired with such a short exposure time, singular value decomposition (SVD) analysis was performed [14-15, 18-20]. SVD is a mathematical technique that factorizes an arbitrary m × n matrix A into the product of three matrices as Eq. II-3 [21].
A = UWVT (Eq. II-3)
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Here U is an m × n column-orthonormal matrix; W is an n × n diagonal matrix of positive singular values, and V is an n × n orthonormal matrix. U and V represent the spectral and positional matrices, respectively. We only retained components of U and V having significantly large singular values to reproduce matrix A, because other components with much smaller singular values contribute to the original data negligibly and can be regarded as noises. Then we reconstructed matrix A by using those components that have significantly large singular values. The number of singular values used for reconstruction depends on the data set, but it was less than 10 in all cases. The SVD was computed in Igor Pro (WaveMetrics) using LAPACK routines.
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Figure II-1. Single colonies on the YES plate after 4–5 days of cultivation at 30 oC.
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Figure II-2. Chemical structures of (a) (R)-(+)-lipoic acid (RLA), (b) glutathione (GSH), (c)
L-ascorbic acid (Vit C), and (d) γ-cyclodextrin (γ-CD).
Figure II-3. Schematic of the laboratory
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Schematic of the laboratory-built confocal Raman microspectrometer.built confocal Raman microspectrometer.
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Figure II-4. The stage-top incubator
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Figure II-5. Estimation of the lateral (XY) resolution of the laboratory-built confocal Raman microspectrometer. Red dots, observed intensity change; blue line, the best fitting result to the model function (Eq. II-1).
Figure II-6. Estimation of the axial (Z) resolution of the laboratory-built confocal Raman microspectrometer. Red dots, observed intensity change; blue line, the best fitting result to the model function (Eq. II-1).
40x103
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Chapter III
Results and Discussion
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III-1. Growth curves
The growth curves of the WT and ∆ppt1 yeast strains in PMLU medium are shown in Figure III-1A. The WT strain can reach stationary phase after 30 h culture. In contrast, the
∆ppt1 strain cannot grow well in PMLU medium primarily due to the deficiency in CoQ10
production. It grows much more slowly than WT and even after 80 h culture, it seems not to reach stationary phase yet. As can be seen from Figure III-1B, however, addition of the antioxidants (RLA, GSH, Vit C, and RLA/γ-CD) was found to improve the growth of the
∆ppt1 mutant.
The cell growth was recovered in all cases, but the extent of recovery depends on antioxidants. The effect is smallest for Vit C. The cell density of the ∆ppt1 strain after 3 days of culture treated with RLA is similar to that of the WT strain, but the growth rate is still much smaller. There is no notable difference in growth curve between RLA and RLA/γ-CD;
the two growth curves look nearly identical. GSH shows the largest recovery in terms of both final cell density and growth rate. Although the ∆ppt1 strain treated with GSH takes somewhat longer time (about 50 h) to reach stationary phase than the WT strain, it indeed eventually grows to the same level as the WT strain. Based on the growth curves, we conclude that the recovery of cell growth increases in the order of Vit C, RLA (RLA/γ-CD), and GSH. This order is thought to reflect, at least to some extent, the strength of antioxidant activity. Similar restoration of the cell growth of another mutant (∆dps1) by addition of CoQ10/γ-CD complex has recently been reported by Nishida et al [22].
III-2. In vivo quantitative Raman assessment of the effects of antioxidants in fission yeast III-2-1. Raman spectra of the WT and ∆∆∆∆ppt1 strains
As shown above, the growth characteristics can clearly show the effect of the antioxidative reagents. However, the cell number does not provide any information on the molecular mechanisms for how the addition of an antioxidant affects the cell physiology of
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the ∆ppt1 strain (e.g., metabolic activity) and results in a better growth. In this study, we used Raman microspectroscopy to understand the effect of antioxidants at the molecular level and to assess their antioxidant activity quantitatively in living cells.
Figure III-2 displays typical Raman spectra of the WT and ∆ppt1 strains. Each spectrum is the average of 200 spectra measured from 200 different cells of either WT or ∆ppt1 strain. It is important to collect data on a large number of cells in order to extract information through statistics that is not affected by cell individuality. The two spectra have been normalized to the area intensity of the 1440 cm−1 band (see Table III-1 for assignment of this band). Because the intracellular space is highly heterogeneous with many organelles and biomolecules, the observed Raman spectral pattern, in general, depends greatly on the location from which the spectrum is measured, namely, where the laser is focused. Here we focused the excitation laser on lipid droplets inside yeast cells (usually visible as tiny black spots under the microscope). Lipid droplets are composed of the core of neutral lipids such as triacylglycerols and sterol esters surrounded by a phospholipid monolayer, so they are rich in lipids.
Several representative Raman bands are observed at 1744, 1655, 1602, 1440, 1300, 1154, and 1003 cm−1 in both WT and ∆ppt1 spectra. The assignments [3] of these and other Raman bands can be found in Table 1. The averaged normalized Raman spectra of the WT and ∆ppt1 strains are quite similar to each other, except for the 1602 cm−1 band. The intensity of this band is much weaker in the ∆ppt1 strain than in WT. As already mentioned in Chapter I, this Raman band is unique in that it sharply reflects the metabolic activity of yeast cells [2, 4, 16], and it has recently been shown to arise mainly from the conjugated C=C stretching of ergosterol [7] (see Figure I-1). Because the ∆ppt1 strain is unable to synthesize CoQ10 by itself, their ability to reduce the level of intracellular reactive oxygen species (ROS) is considered weaker than WT. Note that CoQ10 also has antioxidant activity. In the presence of high levels of ROS, ergosterol can be converted to ergosterol peroxide, and the conjugated
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C=C structure of ergosterol will be disrupted as shown in Figure I-1. Consequently, the intensity of the 1602 cm−1 band will decrease. If this is the case, the intensity of the 1602 cm−1 band should recover when intracellular oxidative stress caused by ROS is reduced by e.g., adding antioxidative reagents externally to the cell.
III-2-2. Raman intensity changes with external addition of antioxidants to the ∆∆∆∆ppt1 strain
To study the effects of exogenous antioxidants on the Raman spectrum of the ppt1 strain, we measured lipid-droplet Raman spectra from 200 living ppt1 cells grown for 10 h in the medium to which the antioxidant (RLA, GSH, Vit C, or RLA/γ-CD) was added. We obtained Raman data exclusively from lipid droplets because their Raman spectrum usually exhibits strong 1602 cm−1 band compared with the cytoplasm. Figure III-3 shows the average of 200 spectra for ∆ppt1 cells with and without the addition of antioxidants. The five averaged spectra have been normalized to the area intensity of the 1440 cm−1 band.
In all cases, the treatment with the antioxidant caused a significant increase in the intensity of the 1602 cm−1 band relative to the control (antioxidant-untreated ∆ppt1). We do not see such a drastic change in other Raman bands. To see this more quantitatively, we plot the number of spectra that give a certain intensity ratio to the 1440 cm−1 band as histograms for the bands at 1602, 1655, 1300, and 1003 cm−1 (Figure III-4–7). These figures compare the distributions of 200 spectra obtained from WT, untreated ∆ppt1 (control), and ∆ppt1 treated with RLA, GSH, Vit C, and RLA/γ-CD. The distributions do not deviate much from the normal distribution. Assuming the normal distribution, we calculated the mean of each distribution and its standard deviation (n = 200), which are also reported in Figure III-4–7.
First, let us focus on the 1602 cm−1 band (Figure III-4). As discussed above, the mean of the intensity ratio A1602/A1440, of the ∆ppt1 strain is about three times smaller than that of WT (0.06 versus 0.18). When treated with RLA, the ∆ppt1 strain shows a mean of 0.12, which is
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double the untreated control value. The mean of the ratios A1602/A1440 is even larger for the
∆ppt1 cells treated with GSH. Upon treatment, it increases by a factor of ≈3.7 and becomes almost the same as WT. In fact, among the four antioxidative reagents tested, GSH showed the most prominent recovery of the 1602 cm−1 band. In contrast, the effect of adding Vit C was found to be little. The trend in the recovery of the band intensity at 1602 cm−1 agrees very well with our growth curve results (see the previous section). The ability of antioxidant to regain the intensity of the 1602 cm−1 band is strongest for GSH, moderate for RLA, and weakest for Vit C. An important point to note here is that using the Raman band at 1602 cm−1, we have been able to assess the activity of antioxidants in vivo and at the molecular level. It is not possible with either growth curve measurements or ordinary biochemical approaches.
We suspected the moderate antioxidant activity of RLA is due to the low solubility of RLA in water, so we also tried a complex of RLA with γ-cyclodextrin (RLA/γ-CD). In the complex, hydrophobic RLA is included in the inner cavity of γ-CD and the outer hydrophilic part makes the guest molecule solubilized in aqueous environment. Nevertheless, we observed no significant difference in the mean value of the intensity ratio A1602/A1440 between treatment with RLA alone and with RLA/γ-CD. This may be because all RLA was dissolved even without using γ-CD complex at concentration as low as 0.5 mM.
The behavior of the protein and lipid Raman bands is quite different from that of the 1602 cm−1 band. The intensity ratios of the 1655 (Figure III-5), 1300 cm−1 (Figure III-6), and 1003 cm−1 (Figure III-7) bands to the 1440 cm−1 band do not vary significantly with antioxidant treatment; the variation is within experimental uncertainties. This result clearly indicates that only the ergosterol band at 1602 cm−1 is sensitive to the treatment with antioxidants and can be used as a probe of the antioxidative effects.
To check the reproducibility of the trend, we performed the whole set of measurements (WT, untreated ∆ppt1, and antioxidant-treated ∆ppt1 strains) in triplicate. More specifically, we used the same excitation laser power and exposure time, and recorded Raman spectra of
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200 cells in each measurement. Results are shown in Figure III-8. The overall trend in the effect of the antioxidants is fully consistent with what we have discussed on the basis of Figures III-4–7.
III-3. Time-lapse Raman measurements
We now have shown by looking into the Raman spectra measured after 10 h of culture, that the intensity of the 1602 cm−1 band of the ∆ppt1 yeast strain can be improved by external addition of the antioxidants (Figure III-3). This phenomenon should be dynamic in nature, and it is crucially important to study its temporal behavior. Therefore, we tried to do time-lapse experiments in which the change in Raman spectrum was monitored at different culture times after the antioxidant was added.
We measured 100 Raman spectra of the ∆ppt1 cells with and without addition of RLA and GSH at 0.5 mM to the medium at different culture times (0, 3, 7, 10, 15, 22, and 30 h). A time-stream of the averaged (but unnormalized) Raman spectra is shown for the control (untreated ∆ppt1), ∆ppt1 treated with RLA, and ∆ppt1 treated with GSH in Figure III- 9, 10, and 11, respectively. At 0 h, the ∆ppt1 strain showed almost no signal at 1602 cm−1 (Figure III-9). The 1602 cm−1 band emerged at 7 h and remains nearly constant for more than 20 h afterward. Note that the intensity of the 1602 cm−1 band slightly increased after long incubation even in the absence of an antioxidant. However, it increased with time more markedly when the ∆ppt1 cells were treated with RLA (Figure III-10) and GSH (Figure III-11). To better see the time evolution, we plot the averaged band intensity ratio A1602/A1440
as a function of culture time (Figure III-12). We can see a drastic increase in the ratio at around 10 h for RLA and GSH. This finding indicates that it took about 10 h for the added antioxidant to become effective. The ratio for the untreated ∆ppt1 strain decayed gradually after 10 h, whereas that for the RLA- or GSH-treated strains increased by about 20–30 times.
In stark contrast, the other Raman bands at 1655, 1300, and 1003 cm−1 did not change with
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time as much as the 1602 cm−1 band (Figure III-13). The marked increase at 10 h can also be seen from histograms of the intensity ratio A1602/A1440 shown in Figure III-14. In both cases of RLA and GSH, the distribution shifts to a higher ratio on going from 3 h to 10 h.
III-4. Conclusion
The key findings are summarized as follows:
(1) Presumably because of the disruption of gene responsible for CoQ10 production, the
∆ppt1 strain of fission yeast may have less resistance to intracellular oxidative stress
∆ppt1 strain of fission yeast may have less resistance to intracellular oxidative stress