Meningitis is an infection of the cerebrospinal fluid CSF around the brain and spinal cord that causes inflammation of the meninges, the protective layers covering the brain.
Meningitis can be caused by viruses, bacteria, or fungi. Although all forms of meningitis are serious, bacterial meningitis is particularly serious. After a 3-hour drive to the hospital, Hannah was immediately admitted.
Physicians took a blood sample and performed a lumbar puncture to test her CSF. They also immediately started her on a course of the antibiotic ceftriaxone, the drug of choice for treatment of meningitis caused by N. Learning Objectives Describe why glycolysis is not oxygen dependent Define and describe the net yield of three-carbon molecules, ATP, and NADH from glycolysis Explain how three-carbon pyruvate molecules are converted into two-carbon acetyl groups that can be funneled into the Krebs cycle.
Glycolysis For bacteria, eukaryotes, and most archaea, glycolysis is the most common pathway for the catabolism of glucose; it produces energy, reduced electron carriers, and precursor molecules for cellular metabolism. Overall, in this process of glycolysis, the net gain from the breakdown of a single glucose molecule is: two ATP molecules two NADH molecule, and two pyruvate molecules. One of the two enzymatic reactions in the energy payoff phase of Embden Meyerhof-Parnas glycolysis that produce ATP in this way is shown here.
Other Glycolytic Pathways When we refer to glycolysis, unless otherwise indicated, we are referring to the EMP pathway used by animals and many bacteria. Transition Reaction, Coenzyme A, and the Krebs Cycle Glycolysis produces pyruvate, which can be further oxidized to capture more energy. In the process, carbon dioxide is released, and one molecule of NADH is formed. Two turns of the Krebs cycle are required to process all of the carbon from one glucose molecule.
The Last Step We have just discussed two pathways in glucose catabolism—glycolysis and the Krebs cycle—that generate ATP by substrate-level phosphorylation. Electron Transport System The electron transport chain ETC or electron transport system ETS is the last component involved in the process of cellular respiration; it comprises a series of membrane-associated protein complexes and associated mobile accessory electron carriers.
There are many circumstances under which aerobic respiration is not possible, including any one or more of the following: The cell lacks genes encoding an appropriate cytochrome oxidase for transferring electrons to oxygen at the end of the electron transport system. The cell lacks genes encoding enzymes to minimize the severely damaging effects of dangerous oxygen radicals produced during aerobic respiration, such as hydrogen peroxide H 2 O 2 or superoxide O2—.
The cell lacks a sufficient amount of oxygen to carry out aerobic respiration. Key Concepts and Summary Glycolysis is the first step in the breakdown of glucose, resulting in the formation of ATP, which is produced by substrate-level phosphorylation ; NADH; and two pyruvate molecules. Glycolysis does not use oxygen and is not oxygen dependent. After glycolysis, a three-carbon pyruvate is decarboxylated to form a two-carbon acetyl group, coupled with the formation of NADH.
The acetyl group is attached to a large carrier compound called coenzyme A. After the transition step, coenzyme A transports the two-carbon acetyl to the Krebs cycle , where the two carbons enter the cycle. The Krebs cycle may be used for other purposes. Many of the intermediates are used to synthesize important cellular molecules, including amino acids, chlorophylls, fatty acids, and nucleotides.
Most ATP generated during the cellular respiration of glucose is made by oxidative phosphorylation. An electron transport system ETS is composed of a series of membrane-associated protein complexes and associated mobile accessory electron carriers. The ETS is embedded in the cytoplasmic membrane of prokaryotes and the inner mitochondrial membrane of eukaryotes.
Each ETS complex has a different redox potential, and electrons move from electron carriers with more negative redox potential to those with more positive redox potential. To carry out aerobic respiration , a cell requires oxygen as the final electron acceptor. A cell also needs a complete Krebs cycle, an appropriate cytochrome oxidase, and oxygen detoxification enzymes to prevent the harmful effects of oxygen radicals produced during aerobic respiration.
Methanogenesis is another form of anaerobic respiration involving the reduction of CO 2. It is discussed in Methanogens and Syntrophy. In aerobic respiration in mitochondria, the passage of electrons from one molecule of NADH generates enough proton motive force to make three ATP molecules by oxidative phosphorylation, whereas the passage of electrons from one molecule of FADH 2 generates enough proton motive force to make only two ATP molecules.
Thus, the 10 NADH molecules made per glucose during glycolysis, the transition reaction, and the Krebs cycle carry enough energy to make 30 ATP molecules, whereas the two FADH 2 molecules made per glucose during these processes provide enough energy to make four ATP molecules. Overall, the theoretical maximum yield of ATP made during the complete aerobic respiration of glucose is 38 molecules, with four being made by substrate-level phosphorylation and 34 being made by oxidative phosphorylation Figure 8.
In reality, the total ATP yield is usually less, ranging from one to 34 ATP molecules, depending on whether the cell is using aerobic respiration or anaerobic respiration and whether the source of electrons is organic or inorganic with the exception of H 2 , generally much less than an organic source. In eukaryotic cells, some energy is expended to transport intermediates from the cytoplasm into the mitochondria, affecting ATP yield. The figure below summarizes the theoretical maximum yields of ATP from various processes during the complete aerobic respiration of one glucose molecule.
Facultative anaerobes are bacteria and archaea that are capable of aerobic respiration, as well as anaerobic metabolism, anaerobic respiration or fermentation. Given the differences in energy yields Table 8.
Table 8. Comparison of energy yields between aerobic and anaerobic respiration. Skip to content 8. Microbial Catabolism. What are the functions of the proton motive force? What are examples of different electron acceptors in anaerobic respiration? Why is anaerobic respiration less energetically favourable than aerobic respiration?
Most ATP generated during the cellular respiration of glucose is made by oxidative phosphorylation. An electron transport system ETS is composed of a series of membrane-associated protein complexes and associated mobile accessory electron carriers.
The ETS is embedded in the cytoplasmic membrane of prokaryotes and the inner mitochondrial membrane of eukaryotes.
Each ETS complex has a different redox potential, and electrons move from electron carriers with more negative redox potential to those with more positive redox potential. To carry out aerobic respiration, a cell requires oxygen as the final electron acceptor. A cell also needs a complete Krebs cycle, an appropriate cytochrome oxidase, and oxygen detoxification enzymes to prevent the harmful effects of oxygen radicals produced during aerobic respiration.
Organisms performing anaerobic respiration use alternative electron transport system carriers for the ultimate transfer of electrons to the final non-oxygen electron acceptors.
Microbes show great variation in the composition of their electron transport systems, which can be used for diagnostic purposes to help identify certain pathogens. The energy of this proton motive force can be harnessed by allowing hydrogen ions to diffuse back through the membrane by chemiosmosis using ATP synthase. Multiple Choice. Fill in the Blank. Short Answer What is the relationship between chemiosmosis and the proton motive force?
How does oxidative phosphorylation differ from substrate-level phosphorylation? How does the location of ATP synthase differ between prokaryotes and eukaryotes? Where do protons accumulate as a result of the ETS in each cell type? Four to five percent of the O 2 consumed by oxidative phosphorylation in the mitochondria is normally converted to reactive oxygen species; therefore defects to the electron transport chain system in cancer cells would result in excessive reactive oxygen species formation [8] — [10].
Ets-1, a member of the Ets protein family of transcription factors, regulates the expression of a diverse set of proteins through its interaction with specific consensus sequences upstream of target genes [16]. The over-expression of Ets-1 has been associated with a multitude of different cancers, specifically with regards to tumour progression and invasion [16] — [19].
Additionally, over-expression of Ets-1 has also been associated with poor prognosis in breast [20] , ovarian [21] , and cervical carcinomas [22]. Traditionally, Ets-1 is thought to function as a transcriptional activator and its high expression in endothelial and stromal cells correlates with tumour cell invasiveness and unfavourable outcome in ovarian and breast cancer [23] — [25].
Our laboratory and that of others have highlighted the importance of Ets-1 in the regulation of different aspects of cancer cell behaviour, including extracellular matrix remodeling, invasion, angiogenesis [26] , and drug resistance [27]. The link between Ets-1 and cancer invasiveness can potentially be explained by the list of known target genes regulated by this transcription factor. Several MMPs and integrin genes, as well as urokinase plasminogen activator uPA , which are all known mediators of extracellular matrix degradation and cell migration, are known targets for Ets-1 [28] — [31].
This transcription factor is thus considered to be an important mediator of cancer cell development and tumour progression. In this study we have demonstrated that Ets-1 plays a role in the regulation of energy metabolism in ovarian cancer cells. Using high throughput genomic analysis, we have found that Ets-1 regulates, either directly or indirectly, several important genes involved in mitochondrial metabolic and antioxidant defense pathways in our Ets-1 over-expression ovarian cancer cell model.
Functionally, we have shown that glycolysis, oxidative phosphorylation, and cellular respiratory systems are altered in response to changes in Ets-1 expression. Taken together, our findings indicate that Ets-1 is a key transcription factor involved in regulating metabolic and oxidative stress in cancer cells.
Ets-1 was over-expressed in ovarian cancer cells using a tetracycline-inducible system, generating Ets1 cells. Ets-1 protein expression in tetracycline-treated and Ets1 was examined via Western blotting Figure 1. Ets-1 protein expression was not detectable in whole cell lysates, but was readily detected in the induced Ets1 lysate. Increased Ets-1 expression was found to be a specific effect as the protein levels of two similar Ets family members, Ets-2 and PEA3 were not altered in this model of Ets-1 over-expression Figure 2.
Western blot analysis showed that neither of these transcription factors was affected by Ets-1 expression in cells. Microarray analysis of and Ets1 cells revealed that 3, genes of the 28, genes probed were up-regulated or down-regulated in response to Ets-1 over-expression by at least 1. For this study, we have chosen to report and examine changes in selected mRNAs whose functions are relevant to mitochondrial activity, cellular metabolism, and oxidative stress.
Real time qRT-PCR validation was conducted using 10 target genes representing various fold change values, and results were normalized to 4 separate housekeeping genes.
Therefore, fold changes greater than 1. We did not find any significant differences in protein expression between and Ets1 cells for any of the enzymes examined data not shown. Microarray analysis of Ets1 ovarian cancer cells revealed that Ets-1 is involved in the regulation of mitochondrial stress and dysfunction as metabolic genes, including those involved in glycolysis, glycolytic feeder pathways, the TCA cycle, and lipid metabolism Table 2 , and genes involved in antioxidant defense Table 3 were altered in Ets-1 over-expressing cells.
To evaluate whether cells with stable over-expression of Ets-1 favour glycolysis over oxidative phosphorylation for energy, as predicted by our microarray analysis, cells were grown in glucose-free media supplemented with the glycolytic inhibitor 2-DG, an analog of glucose.
Cells were grown in the presence of varying amounts of 2-DG, and representative growth curves were generated for each cell line. The growth of cells in media containing 2-DG was inhibited to a greater extent in cells with a higher Ets-1 expression than in parental cells Figure 3A. Our results indicated that Ets1 cells induced with tetracycline were the most sensitive to 2-DG, with an IC 50 of 0. To discount any treatment effect, parental cells were treated with tetracycline, and showed no significant growth inhibition by 2-DG with an IC 50 of 3.
Growth of cells in normal or glucose-free media supplemented with sodium pyruvate was compared over 96 hrs, after which it was observed that the proliferation of cells with increased expression of Ets-1 was notably slower compared to parental cells Figure 3B.
Treatment with glucose-free media resulted in a decreased proliferation rate for all cells tested in comparison to normally supplemented media data not shown. B and Ets1 cells were grown in the absence of glucose, and proliferation assays were conducted at 24 hour intervals. Our microarray analyses suggest that Ets-1 over-expression resulted in an overall down-regulation of genes that encode electron transport chain components, suggesting that these cell lines would likely display decreased O 2 consumption Table 4.
This assumption was evaluated using high-resolution respirometry, where basal oxygen consumption was measured following the addition of cells to an oxygraph.
Basal oxygen consumption was significantly lower in induced Ets1 cells No significant tetracycline treatment effect on basal oxygen consumption was found following induction of cells, confirming that tetracycline did not affect oxygen consumption in our model data not shown. Basal oxygen consumption was determined for and Ets1 ovarian cancer cells. Ets-1 expression was associated with a significant decrease in basal oxygen consumption suggesting a decreased reliance on oxidative phosphorylation in Ets1 cells.
Mitochondrial reactive oxygen species activate several key signaling pathways involved in tumourigenesis and up-regulate the expression of important oncogenic transcription factors including Ets-1 [32].
Subsequent treatment of these cells with H 2 O 2 increased Ets-1 expression in a dose-dependent manner, suggesting that this transcription factor is highly responsive to tumour-derived signals. Our previous analysis of the Ets-1 promoter led to the identification of an antioxidant response element ARE that proved to be pivotal in regulating the expression of Ets-1 under both basal and H 2 O 2 -induced conditions [15]. Traditionally, the functional importance of Ets-1 over-expression in cancer has been associated with the regulation of matrix-degrading proteases and angiogenic factors [26] , [34].
However, our recent findings that mitochondrial reactive oxygen species potently affects Ets-1 expression at the transcriptional level [15] suggest that the importance of this transcription factor in cancer initiation and progression extends beyond angiogenesis and metastasis alone. We hypothesized that Ets-1 may be involved in the regulation of mitochondrial metabolism in cancer cells because mitochondrial stress from both increased reactive oxygen species production and electron transport chain malfunction result in increased Ets-1 mRNA and protein [15].
In order to determine the functional importance of Ets-1 expression in cancer cell metabolism, we generated the tetracycline-inducible Ets-1 over-expressing ovarian cancer cell line Ets1. Parental cells do not express detectable levels of Ets-1 protein endogenously. To analyze the genomic consequences of Ets-1 over-expression in these ovarian cancer cells, we conducted a human gene microarray.
Our findings indicate that Ets-1 is either directly or indirectly involved in regulating the expression of more than 3, of the over 28, human genes examined.
Interestingly, our findings suggest that Ets-1 could act as a transcriptional repressor of more than half 1, of these genes in ovarian carcinoma cells.
Although Ets-1 has been studied extensively in both physiological and pathological processes [17] , it is rarely referred to as a transcriptional repressor. In the context of mitochondrial dysfunction and metabolism, Ets-1 was found to at least partially down-regulate several components of the electron transport chain. Complex I, the most prominent site for electron leakage leading to excessive reactive oxygen species production in the electron transport chain, is composed of 39 nuclear encoded subunit genes, of which Ets-1 down-regulates Another important site for electron leakage and reactive oxygen species production is Complex III, which is composed of 10 subunits, of which Ets-1 down-regulates 3.
Taken together, these repressive functions suggest that Ets-1 is prominently involved in the decreased reliance on oxidative phosphorylation frequently associated with cancer cells. Consequently, genes encoding mitochondrial proteins involved in oxidative phosphorylation would be down-regulated, and cells would require alternate methods of ATP production including glycolysis and fatty acid oxidation.
Given that components of almost every complex of the electron transport chain, several key enzymes of the TCA cycle, and ultimately the reducing equivalents needed for electron transport were similarly down-regulated, Ets-1 over-expressing cells appear to have a decreased capacity to generate ATP via oxidative phosphorylation at the gene expression level.
Cancer cells commonly have a decreased reliance on oxidative phosphorylation for energy generation, and they likewise have an increased dependence on glycolysis and fat metabolism for cellular energy [35]. In further support that Ets-1 is involved in the regulation of altered cancer metabolism, Ets-1 is associated with the increased expression of many genes involved in glycolysis, glycolytic feeder pathways, and the pentose phosphate pathway.
Additionally, the expression of Ets-1 is also correlated with increases in the expression of many genes involved with lipid metabolism and biosynthesis. Taken together, these trends suggest that Ets-1 is an important transcriptional regulator not only in the catabolic, but also anabolic metabolic transitions of cancer cells that ultimately promote tumourigenesis [35]. It was almost half a century ago when the up-regulation of fatty acid synthase FASN was first described in cancer [36] , which is now known to be over-expressed in the majority of cancers.
Interestingly, Ets-1 was also found to be involved in the regulation of increased FASN gene expression in our ovarian cancer model. Thus, our gene expression findings suggest that Ets-1 is a key transcription factor involved in the metabolism of cancer cells, and particularly important in the metabolic shift towards glycolysis and anabolic means of energy production. Although it is important to note that Etsmediated metabolic regulation is likely achieved by a large consortium of different transcription factors, a more complex regulatory network of transcription factors influenced by mitochondrial dysfunction have yet to be elucidated.
We have demonstrated that the over-expression of Ets-1 in our ovarian cancer model did not affect protein levels of two closely related ETS family members; however, it is possible that other ETS transcription factors from this very large family also influence cancer metabolism. Considering that these transcription factors recognize almost identical consensus sequences, the repetition of the experiments within this study following the over-expression of other ETS family members could yield similar results.
In addition, such experiments would further characterize the potentially large transcriptional network involved in the specialized metabolism of cancer cells. To determine the functional relevance of our genomic results, we have examined the glycolytic capability of our Ets-1 over-expression model. We have indirectly evaluated the oxidative phosphorylation capacity of these cells through treatment with the glycolytic inhibitor 2-DG. Additionally, the Ets-1 over-expressing ovarian cancer cells displayed significantly decreased growth following glucose deprivation, further emphasizing their glycolytic reliance.
The growth rate of all cell types in glucose-free media was decreased as compared to normally supplemented media, particularly after 48 hrs when a distinct divergence in cell growth was consistently observed, likely due to decreased glucose availability.
Over-expression of Ets-1 potently exacerbated this divergence as the growth of induced Ets1 cells drastically decreased after 48 hrs. However at the protein level, we did not find any significant differences in the expression of glucosephosphate dehydrogenase, pyruvate dehydrogenase, cytochrome c , or hexokinase, which are all enzymes involved in glycolysis or oxidative phosphorylation. Importantly, we did see repeatable and significant differences in glycolytic dependence associated with Ets-1 expression, and the lack of changes in protein expression could therefore be due to post-translational modifications mediated by Ets For example, differences within the catalytic region of an enzyme would result in functional differences, but would not necessarily be detectable via Western blot, as this technique is dependent on the specific epitope targeted by the antibody used.
Thus, we plan to examine the enzyme activity of these metabolic enzymes in future studies to determine if any differences exist between and Ets1 cells that could account for the functional differences we have demonstrated in this study. The evaluation of O 2 consumption of a population of cells is a functional evaluation of oxidative capacity, as it represents a good estimate of the rate at which electrons are passing along the electron transport chain and being reduced to H 2 O 2.
The polarographic system used in this study to measure O 2 consumption includes sensors that yield a current proportional to the partial pressure of O 2 in cell containing media by consuming O 2 in a cathode half reaction, thus the signal responds exponentially to changes in O 2 pressure within the sample. In the absence of specific complex substrates and ADP, thereby simulating respiration, the basal rate of O 2 consumption can be measured.
We have observed significant decreases in O 2 consumption in cells with increased Ets-1 expression. Our results indicate that Ets-1 is directly involved in the regulation of cellular oxidative capacity, where Ets-1 over-expression led to significantly decreased O 2 consumption, and Ets-1 down-regulated cells displayed a very prominent increase in O 2 consumption.
These results suggest that up-regulated Ets-1 expression promotes a decreased dependence on oxidative phosphorylation for energy, and provides further evidence towards the functional importance of Ets-1 in cancer cell metabolism. High levels of oxidative stress are typically observed in the tumour microenvironment as a result of imbalances in antioxidant defense factors, and impaired DNA repair mechanisms [37] — [39].
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