Chapter III Results and Discussion
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
induced by ROS. It showed much weaker 1602 cm−1 band intensity than the WT strain (Figure III-2).
(2) Adding antioxidants to the medium in which the ∆ppt1 strain was grown recovered the intensity of the 1602 cm−1 band (Figure III-4). However, similar effects of the antioxidants were not observed for other Raman bands at 1655, 1300, and 1003 cm−1 (Figures III-5-7).
(3) The antioxidant activity in living fission yeast cells evaluated by using the diminished 1602 cm−1 band of ∆ppt1 was found to be strongest for GSH (about 2.8-fold increase with respect to the untreated control), moderate for RLA and RLA/γ-CD (about 2-fold increase), and very weak for Vit C (1.2-fold increase).
(4) Time-lapse Raman measurements on the ∆ppt1 cells treated with RLA and GSH clearly revealed that the intensity of the 1602 cm−1 band begins to increase approximately 10 h after the addition and keeps growing up to 30 h.
Together, we have demonstrated that Raman microspectroscopy can be used as a novel quantitative tool for assessing the efficacy of various antioxidants in vivo. It is still unclear why only the ergosterol band recovers when cells are treated with antioxidants and other protein and lipid bands do not. More spectroscopic as well as biological experiments will be needed to clarify the molecular mechanism behind the present findings.
24
Table III-1. Assignments [3] of representative vibrational bands observed in the Raman spectrum of S. pombe
.
Peak position (cm−1) Assignment
715 Phospholipid headgroup
782 DNA/RNA
1003 Ring breathing of phenylalanine residues
1083 Antisymmetric CCC stretching
1154 C–C and C–N stretching
1266 C=C–H in-plane bend of the cis –CH=CH– linkage Amide III mode of proteins
1300 In-plane CH2 twisting mode
1340 CH2 bending of the aliphatic chain of proteins 1440 CH2 scissoring and CH3 degenerate deformation 1602 Mainly conjugated C=C stretch of ergsterol [9]
1655 cis-C=C stretching of the unsaturated lipid chains Amide I mode of proteins
1744 C=O stretch of the ester linkage of lipids
25
Figure III-1. (A) Growth curves of the WT (black) and untreated ∆ppt1 strains (red). (B) growth curves of the untreated ∆ppt1 strain (red) and that treated with RLA (green), GSH (blue), Vit C (purple), and RLA/γ-CD (orange).
6.5
log(No. of cells/ 1 mL)
80
26
Figure III-2. Averaged Raman spectra (n = 200) of the WT (black) and ∆ppt1 strain (red).
The spectra have been normalized to the area intensity of the 1440 cm−1 band.
1200 1000 800 600 400
Intensity
1800 1600 1400 1200 1000 800 600
Raman shift (cm-1)
ppt1
WT
∆
17441655 1602
1440
1300
1003
27
Figure III-3. Averaged Raman spectra (n = 200) of the untreated ∆ppt1 strain (red) and the
∆ppt1 strains treated with 0.5 mM RLA (green),0.5 mM GSH (blue), 0.5 mM Vit C (purple), and 0.5 mM RLA/γ-CD (orange).
2000 1800 1600 1400 1200 1000 800 600 400
In te ns it y
1800 1600 1400 1200 1000 800 600 Raman shift (cm-1)
ppt1 RLA GSH Vit C RLA/ CD γ
∆
28
Figure III-4. Histograms of the ratio of the area intensity of the 1602 cm−1 band to the 1440 cm−1 band, A1602/A1440. The number of bins is 20 and the bin width is 0.02. Yellow lines
29
Figure III-5. Histograms of the ratio of the area intensity of the 1655 cm−1 band to the 1440 cm−1 band, A1655/A1440. The number of bins is 20 and the bin width is 0.03. Yellow lines
30
Figure III-6. Histograms of the ratio of the area intensity of the 1300 cm−1 band to the 1440 cm−1 band, A1300/A1440. The number of bins is 20 and the bin width is 0.025. Yellow lines
31
Figure III-7. Histograms of the ratio of the area intensity of the 1003 cm−1 band to the 1440 cm−1 band, A1003/A1440. The number of bins is 20 and the bin width is 0.005. Yellow lines
32
A.
B.
C.
D.
Figure III-8. Bar graphs of the averaged intensity ratio of the 1602 (A), 1655 (B), 1300 (C), and 1003 (D) cm−1 bands to the 1440 cm−1 band.
WT Dppt1 RLA GSH Vit C RLA/gCD
Ratio to 1440 cm-1
bandarea of 1602 cm-1
***P<0.001
WT Dppt1 RLA GSH Vit C RLA/gCD
Ratio to 1440 cm-1
bandarea of 1655 cm-1
***P<0.001
WT Dppt1 RLA GSH Vit C RLA/gCD
Ratio to 1440 cm-1 bandarea of 1300 cm-1***P<0.001
0.000
WT Dppt1 RLA GSH Vit C RLA/gCD
Ratio to 1440 cm-1
bandarea of 1003 cm-1
***P<0.001
33
Figure III-9. Time-lapse Raman spectra (n =100) of the untreated ∆ppt1 strain obtained 0 h (red), 3 h (orange), 7 h (green), 10 h (blue), 15 h (purple), 22 h (mulberry), and 30 h (brown).
2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0
R am an I nt en si ty
1800 1600 1400 1200 1000 800 600 Raman shift (cm-1)
30 h 22 h 15 h 10 h 7 h 3 h 0 h without antioxidant
34
Figure III-10. Time-lapse Raman spectra (n = 100) of the ∆ppt1 strain treated with 0.5 mM RLA obtained 0 h (red), 3 h (orange), 7 h (green), 10 h (blue), 15 h (purple), 22 h (mulberry), and 30 h (brown) after addition of RLA.
2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0
R am an i nt en si ty
1800 1600 1400 1200 1000 800 600
Raman shift (cm
-1)
0 h
3 h
7 h
10 h
15 h
22 h
30 h
add 0.5 mM RLA
35
Figure III-11. Time-lapse Raman spectra (n = 100) of the ∆ppt1 strain treated with 0.5mM GSH obtained 0 h (red), 3 h (orange), 7 h (green), 10 h (blue), 15 h (purple), 22 h (mulberry), and 30 h (brown) after addition of GSH.
2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0
R am an i nt en si ty
1800 1600 1400 1200 1000 800 600
Raman shift (cm
-1)
0 h
3 h
7 h
10 h
15 h
22 h
30 h
add 0.5 mM GSH
36
Figure III-12. Time dependence of the averaged ratio of Raman band area intensity at 1602 cm−1 to that at 1440 cm−1 of the untreated ∆ppt1 (red), ∆ppt1 treated with 0.5 mM RLA (green), and ∆ppt1 treated with 0.5 mM GSH (blue).
0.25
0.20
0.15
0.10
0.05
0.00
ratio
30 25
20 15
10 5
0
Time (hr) A1602/A1440 (add GSH)
A1602/A1440 (add RLA)
A1602/A1440 (without antioxidant)
37
Figure III-13. Time dependence of the averaged ratio of Raman band area intensity at 1655 (triangle), 1300 (circle), and 1003 (square) cm−1 to that at 1440 cm−1 of the untreated ∆ppt1 (red), ∆ppt1 treated with 0.5 mM RLA (green), and ∆ppt1 treated with 0.5 mM GSH (blue).
0.5
0.4
0.3
0.2
0.1
0.0
ratio
30 25
20 15
10 5
0
Time (hr)
A1655/A1440 (non-add) A1655/A1440 (add GSH) A1655/A1440 (add RLA) A1300/A1440 (non-add) A1300/A1440 (add GSH) A1300/A1440 (add RLA) A1003/A1440 (non-add) A1003/A1440 (add GSH) A1003/A1440 (add RLA)
38
A
B
Figure III-14. Histograms of the ratio of Raman band area intensity at 1602 cm−1 to that at 1440 cm−1 3 h (A) and 10 h (B) after addition of the antioxidant (green, RLA; blue, GSH).
Yellow lines represent the mean value.
60
39
Chapter IV
Summary and Future Work
40
In this study, we used confocal Raman microspectroscopy to measure the effects of exogenous antioxidants on the CoQ-deficient (∆ppt1) strain of fission yeast. The Raman intensity of the 1602 cm−1 band, which mainly originates from the conjugated C=C stretch of ergosterol [9], is greatly suppressed in the ∆ppt1 strain. This is most likely because the ∆ppt1 strain cannot lower the level of ROS and ergosterol can potentially be oxidized by accumulated ROS to form ergosterol peroxide [12, 13], which no longer possesses the conjugated C=C moiety. Upon addition of antioxidants (RLA, GSH, Vit C, and RLA/γCD), the intensity of the 1602 cm−1 band increased about 2–3 times. However, similar recovery of the band intensity was not observed for other Raman bands at 1655, 1300, and 1003 cm−1, showing that the 1602 cm−1 band is a sensitive probe for intracellular oxidative stress.
Time-lapse Raman measurements on the ∆ppt1 cells treated with RLA and GSH clearly revealed that the intensity of the 1602 cm−1 band begins to increase approximately 10 h after the addition and keeps growing up to 30 h.
However, in these experiments, we did not discuss the effect of the concentration of exogenous antioxidants on the CoQ-deficient (∆ppt1) strain of fission yeast, so we will measure the CoQ-deficient (∆ppt1) strain of fission yeast added different concentration antioxidants. Time-lapse Raman measurements on the ∆ppt1 cells treated with RLA and GSH clearly revealed that the intensity of the 1602 cm−1 band begins to increase approximately 10 h after the addition and keeps growing up to 30 h. But the intensity of the 1602 cm-1 did not keep a balance, so we will observes the intensity of the 1602 cm-1 after 30 h.
We have thus demonstrated that Raman microspectroscopy can be used as a novel quantitative tool for assessing the efficacy of various antioxidants in vivo. Although it is still unclear why only the ergosterol band recovers when cells are treated with antioxidants and other protein and lipid bands do not, we believe this method will open new possibilities not only in fundamental cell biology but in medicine and food industry.
41
Reference
42
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