1. Stephanie P. Cartwright, Roslyn M. Bill*, Alan R. Hipkiss Aston Research Centre for Healthy Ageing, School of Health and Life Sciences, Aston University, Birmingham, United Kingdom Abstract 指導者 : 藍清隆 教授 演講者 : 俞曉雯 1

2.  什麼是 Dipeptide L-Carnosine?  學名是 β- 丙氨醯 -L- 組氨酸 (β-alanyl-L-histidine) ， 是一種由 β- 丙氨酸和 L- 組氨酸兩種胺基酸組成 的二肽，存在於腦部、腎臟以及骨骼肌組織。  具有很強的抗氧化能力，對人體有益。  已被證實可清除活性氧自由基 (ROS) 、 α-β 不飽 和醛。  可改善因曝露在胍下導致大鼠水晶體混濁而產 生的白內障。 2

3.  What are L-carnosine able to do?  # delay cell senescence  # reverse the senescent fibroblasts  # extending lifespan in Drosophila melanogaster  # selectively inhibits transformed cell lines & other tumor cells 3

4.  Warburg effect( 瓦氏效應 )  由奧托 · 海因里希 · 瓦爾堡 (Otto Heinrich Warburg) 所提出的理論，認為癌細胞的生長 速度遠大於正常細胞的原因來自於能量的來 源差別。癌細胞會偏向使用醣解作用取代一 般正常細胞的有氧循環，所以癌細胞使用粒 線體的方式與正常細胞就會有所不同。 4

5.  Crabtree effect  The inhibition of respiration in the presence of glucose 5

6.  When Saccharomyces cerevisiae grow on a fermentable carbon source(2% glucose), aerobic respiration is actively down-regulated.  S.cerevisiae grow on non-fermentable carbon sources, such as 2% glycerol(by employing oxidative phosphorylation for ATP generation.) 6

7.  Why was S. cerevisiae chosen as a test system?  L-carnosine slows growth rates and increases cell death of yeast cells when they are reliant on glycolysis for energy generation.When yeast is reliant on oxidative phosphorylation, it is resistant to L-carnosine’s inhibitory effects. 7

8.  ●Yeast Strains and Culturing Conditions  YP medium: 1% yeast extract, 2% bacto peptone and 2% of the desired carbon source  2xCBS: 10 g/L ammonium sulfate, 6 g/L potassium dihydrogen phosphate, 1 g/L magnesium sulfate heptahydrate supplemented with 2% glucose.  2xDO solution: 100 mM MES, 2 mL/L each of trace element solution and vitamin stock solution.  10xDO solution(per L): 200 mg adenine hemisulfate, 200 mg L- arginine hydrochloride,200 mg L-histidine hydrochloride monohydrate, 300 mg L-isoleucine,1000 mg L-leucine, 300 mg L-lysine hydrochloride,200 mg L-methionine,500 mg L-phenylalanine, 2000 mg Lthreonine,200 mg L-tryptophan,300 mg L-tyrosine, 200 mg uracil, 1,500 mg L-valine. 8

9.  250 mL trace element solution: 3.75 g EDTA,1.125 g zinc sulfate heptahydrate,0.25 g magnesium chloride tetrahydrate,0.075 g colbalt (II) chloride hexahydrate,0.075 g copper (II) sulfate pentahydrate,0.1 g sodium molybdenum dehydrate,1.125 g calcium chloride dehydrate,0.75 g iron (II) sulfate heptahydrate, 0.25 g boric acid and 0.025 g potassium iodide.  250 mL vitamin stock solution: 0.0125 g biotin,0.25 g calcium-D-antothenate,0.25 g nicotinic acid,6.25 g myo- inositol,0.25 g thiamine hydrochloride,0.25 g pyridoxine hydrochloride and 0.05 g Damino benzoic acid. 9

10.  How to initiate the experiment?  5 mL YP or 2xCBS supplemented with 2% glucose were inoculated with a single yeast colony, grown to logarithmic phase and used to inoculate a 125 mL baffled shakeflask containing 25 mL growth medium. Cultures were incubated at 30°C,220 rpm.  L-carnosine,  -alanine,L-histidine and D- carnosine were prepared to use. 10

11.  ●Sampling and Extracellular Substrate Determination 1.Glucose concentrations were calculated 2.Ethanol analysis was performed 3.To determine cell viability  ●On-line Flow Microcalorimetry 11

12.  ●L-Carnosine Decreases the Specific Growth Rate of Yeast Cells Grown on the Fermentable Carbon Source, 2% Glucose  In exponential growth, respiration is inhibited  Table 1: 1.10 mM,20 mM and 30 mM Lcarnosine 2.10 mM D-carnosine or 10 mM  -alanine or L-histidine 3.30 mM b-alanine and 30 mM L-histidine 12

13. 13

14.  ●L-Carnosine Reduces the Viability of Glucose-Grown Cells  Figure 1 Figure 1  Table 1 Table 1  10% in 10 mM L-arnosine V.S 17% in 30 mM Lcarnosine (Table 1)  10 mM Dcarnosine & 10–30 mM b-alanine or L-histidine 14

15.  ● L-Carnosine Affects the Metabolism of Glucose-grown Cells  Figure 2 Figure 2  Why Microcalorimetry do not fit strictly to the exponential curve 1.OD 600 :measure cell’s density 2.Heat output:measure the metabolically active. 15

16.  ●L-Carnosine Increases the Specific Growth Rate of Cells Grown on the Non-fermentable Carbon Source, 2% Glycerol, and does not Reduce their Viability  Table 1 Table 1 16

17.  ●The Metabolism-dependent Effects of L- carnosine are Observed for Cells Grown on a Range of Carbon Sources  Fermentable V.S Non-fermentable carbon sources  Table 2 Table 2  ●The Respiratory Yeast, Pichia pastoris, is Resistant to Lcarnosine-induced Cell Death 17

18.  ● Yeast Strains with Deletions in Nutrient-sensing Pathways are Resistant to L-carnosine-induced Cell Death  Table 3 Table 3 18

19. 19

20. 20

21. 21

22.  L-Carnosine 1.Increase in the lifespan 2.Show the effect on yeast 3.Inhibits the growth of cancer cells  Crabtree effect  Table 2 Table 2 22

23.  non-fermentable carbon sources require the presence of oxygen to be metabolized via oxidative phosphorylation.  Table 1 Table 1  L-carnosine has a detrimental effect on cells when their energy generation is dependent on glycolysis. 23

24.  The data for yeast cells grown on glucose are consistent with observations previously published for cancer cells  What do yeast cells have?  on glucose inhibited on glycerol not inhibited  D-carnosine does not affect growth on glucose, but significantly stimulates growth on glycerol 24

25.  1. 搜尋引擎：維基百科，搜尋字樣： Carnosine ，搜尋網址： http://zh.wikipedia.org/wiki/%E8%82%8C%E8%82%BD ， 2012/11/25 。  2. 搜尋引擎：維基百科，搜尋字樣： Warburg effect ，搜尋網址： http://zh.wikipedia.org/zh- tw/%E7%93%A6%E6%B0%8F%E6%95%88%E6%87%89#cite_note-1 ， 2012/11/25 。  3. 搜尋引擎：維基百科，搜尋字樣： Crabtree effect ，搜尋網址： http://en.wikipedia.org/wiki/Crabtree_effect ， 2012/11/25 。 25