Passage
Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H₂O₂, superoxide (O₂⁻) , and hydroxyl radicals (^•OH) interact destructively with biological molecules. Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space. Researchers incubated cultured rat cells (PC12 cells) with H₂O₂ in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue. Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity. After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H₂O₂ and with varying concentrations of GAH for 4 hours. Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1) .
Figure 1 Extracellular LDH activity from PC12 cells exposed to H₂O₂ and varying concentrations of GAH. p-values are relative to 5 mM H₂O₂ and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H₂O₂ and varying concentrations of GAH (Figure 2) .
Figure 2 Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H₂O₂ in the presence and absence of 1 mg/mL GAH. Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells. The percentage of apoptotic cells in each condition is shown in Figure 3.
Figure 3 Percentage of apoptotic cells in the presence or absence of H₂O₂ and GAH
-What is the lowest tested concentration of GAH that produces a significant reduction in extracellular LDH activity when 5 mM H₂O₂ is used?

Question

Passage
Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H₂O₂, superoxide (O₂⁻) , and hydroxyl radicals (^•OH) interact destructively with biological molecules. Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space. Researchers incubated cultured rat cells (PC12 cells) with H₂O₂ in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue. Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity. After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H₂O₂ and with varying concentrations of GAH for 4 hours. Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1) .

Passage Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H<sub>2</sub>O<sub>2</sub>, superoxide (O<sub>2</sub><sup>−</sup>) , and hydroxyl radicals (<sup>•</sup>OH) interact destructively with biological molecules. Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space. Researchers incubated cultured rat cells (PC12 cells) with H<sub>2</sub>O<sub>2</sub> in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue. Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity. After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H<sub>2</sub>O<sub>2</sub> and with varying concentrations of GAH for 4 hours. Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1) . <strong>Figure 1</strong> Extracellular LDH activity from PC12 cells exposed to H<sub>2</sub>O<sub>2</sub> and varying concentrations of GAH. p-values are relative to 5 mM H<sub>2</sub>O<sub>2</sub> and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H<sub>2</sub>O<sub>2</sub> and varying concentrations of GAH (Figure 2) . <strong>Figure 2</strong> Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H<sub>2</sub>O<sub>2</sub> in the presence and absence of 1 mg/mL GAH. Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells. The percentage of apoptotic cells in each condition is shown in Figure 3. <strong>Figure 3</strong> Percentage of apoptotic cells in the presence or absence of H<sub>2</sub>O<sub>2</sub> and GAH -What is the lowest tested concentration of GAH that produces a significant reduction in extracellular LDH activity when 5 mM H<sub>2</sub>O<sub>2</sub> is used? A) 0.0125 mg/mL B) 0.0625 mg/mL C) 0.25 mg/mL D) 1 mg/mL

Figure 1 Extracellular LDH activity from PC12 cells exposed to H₂O₂ and varying concentrations of GAH. p-values are relative to 5 mM H₂O₂ and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H₂O₂ and varying concentrations of GAH (Figure 2) .

Figure 2 Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H₂O₂ in the presence and absence of 1 mg/mL GAH. Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells. The percentage of apoptotic cells in each condition is shown in Figure 3.

Figure 3 Percentage of apoptotic cells in the presence or absence of H₂O₂ and GAH
-What is the lowest tested concentration of GAH that produces a significant reduction in extracellular LDH activity when 5 mM H₂O₂ is used?

Quizplus · Accepted Answer

11ecba2d_0f1c_4c0d_a3bf_df60cd51a668_MD0008_00 p-values are expressed as decimals between 0 and 1, and measure the probability that a difference between two data sets is due to random chance rather than two truly different results. For example, a p-value of 0.5 indicates a 50% chance that an observed difference is merely due to random chance. Lower p-values indicate a lower probability of random chance, and therefore a higher probability that the observed difference is real (ie, due to experimental differences). Generally, a difference is accepted as statistically significant if p < 0.05 (ie, 95% chance of a true difference). Figure 1 shows extracellular LDH activity for several concentrations of GAH, as well as their p-values relative to 5 mM H₂O₂ in the absence of GAH. Of the GAH concentrations tested, 0.25 mg/mL is the lowest concentration that yields a p-value of less than 0.05 (p = 0.009). Therefore, 0.25 mg/mL is the lowest tested concentration that produces a significant reduction in extracellular LDH activity. (Choice A) 0.0125 mg/mL of GAH yields nearly the same extracellular LDH activity as 0 mg/mL and produces a p-value of 0.81. For a difference to be statistically significant, the p-value must be less than 0.05. (Choice B) 0.0625 mg/mL of GAH leads to an apparently lower extracellular LDH activity than when no GAH is added, but the data produce a p-value of 0.067. For a difference to be statistically significant, the p-value must be less than 0.05. (Choice D) 1 mg/mL of GAH produces a significant reduction in oxidative stress (p = 0.002), but the question asks for the lowest tested dose that produces a significant response. 0.25 mg/mL also produces a significant reduction in oxidative stress, so 1 mg/mL is not the lowest dose that yields a significant response. Educational objective: p-values are statistical measures of the probability that an observed difference is due to random chance. Low p-values indicate a low probability that a difference between two observations is a coincidence. Generally, p-values of less than 0.05 (5% chance of coincidence) are accepted as statistically significant.

Passage Oxidative Stress Occurs in Cells When Reactive Oxygen Species (ROS)