Ammk1 Week 1 Lab Report

The first enzyme marker used was Succinate Dehydrogenase which catalyses the oxidation of succinate to fumarate in the tricarboxylic acid cycle (Hollywood, et al., 2010). This cycle occurs within mitochondria and succinate dehydrogenase is specifically found within the inner mitochondrial membrane. Therefore, succinate dehydrogenase activity is an indicator of mitochondrial activity (Berg, et al., 2007). Mitochondria are large organelles, approximately 0.5-1m in diameter and 5-10m in length with a high

Mitochondria, dubbed the ‘powerhouse of the cell’, are a type of organelle present in most human cells. Their primary function is to generate Adenosine Triphosphate (ATP), the cell’s principal source of chemical energy. Unlike most other organelles, mitochondria store their own set of genetic material, distinct from the DNA situated in a cell’s nucleus. Although this ‘mitochondrial genome’ represents only 0.1% of a cell’s genetic information, it often plays a significant role in development.

Under normal conditions, the mitochondria maintain cytosolic Ca2+ levels, which is necessary for normal cellular function. However, the mitochondrial uptake of excessive levels of Ca2+ can lead to inhibition of ATP synthesis, disruption of mitochondrial membrane potential, increased ROS production, and generation of the mitochondrial permeability transition (mPT) state, which is thought to occur in response to formation of the mPT pore. As a consequence, cellular demise can occur through necrotic-related mechanisms events, including a loss of energy production and oxidative stress as well as apoptotic-related mechanisms, including the mitochondrial release of pro-apoptotic proteins. Given the central role of the mitochondria in cell

(8). Our preliminary data indicate that alveolar type (ATII) cells isolated from individuals with emphysema have higher nuclear DSBs than control smokers or nonsmokers. Moreover, we observed an increase in mtDNA damage in ATII cells in this disease in comparison with controls. We also found lower XRCC4-like factor (XLF) expression, which is involved in NHEJ pathway (9, 10), in ATII cells in emphysema in comparison with controls. Furthermore, we detected that high oxidative stress induced by exposure to cigarette smoke induces XLF oxidation and localization in mitochondria. DJ-1 is a cytoprotective protein localized in mitochondria. However, we observed that it interacts with XLF in ATII cells in emphysema, which indicates the critical role of XLF/DJ-1 complex in mitochondrial function. In addition, our results suggest that the number of mitochondria is decreased in these cells isolated from emphysema patients in comparison with control smokers and nonsmokers. Our hypothesis is that high levels of ROS in emphysema induce XLF oxidation and mtDNA damage leading to mitophagy and cell death (Fig. 1). Elucidating the molecular mechanisms contributing to mitophagy in primary ATII cells will advance our understanding of the contribution of mitochondria physiology to emphysema development. ATII cells will be isolated from excess tissue obtained from lung transplants of patients with emphysema, Veterans with respiratory problems and from control organ donors

Presbycusis or Age-related hearing impairment is the most frequent sensory disorder in the adults. It affects more than half of all adults by age seventy-five years. Presbycusis is an irreversible symmetrical progressive loss of hearing sensitivity during the lifespan. It has an insidious onset without clinical manifestation so detected by the routine diagnostic test after losing or damaging to inner ear cells and neurons. Presbycusis counted as an untreatable disorder since these inner ear structures lose the ability of regeneration. Mitochondria by production reactive oxygen species or inducing apoptotic genes is one of most prominent intracellular organelles that contribute in presbycusis progression. The present study focused on mitochondrial

In the current work, activation of MEK by doxycycline induction led to an increase in functional MEK protein production in a dose-dependent way. Our results depict that continuous MEK activation in TT-MEK cells inhibited tumor cell growth. Notably, this growth reduction was dependent on the levels of the MEK protein present. We have also demonstrated that activation of MEK in MTC cells reduces the levels of CgA and ASCL1. These findings provide the first documentation of the role of MEK signaling as a tumor suppressor in MTC cells.

Mitochondrial damage is a normal part of aging, but is accelerated in many metabolic disorders. Chronic deficiencies and gut imbalances can destroys the mitochondrial membranes and lead to the modern diseases we see today.

This results in an increase in appetite and stimulation of catabolic activity, such as glycolysis (Lim et al., 2013). However, it causes a decline in AMPK activity in the liver and adipose tissue, which can lead to inhibition of anabolic processes, such as lipogenesis (Kola et al., 2005; Lim et al., 2013). AMPK also plays a key role in cellular energy homoeostasis in relation to the opposing effects of leptin and ghrelin in the hypothalamus (Kola et al., 2005). Moreover, the enzyme, AMPK, is involved in a major pathway associated with the regulation of appetite and metabolism (Kola et al.,

Figure 9: Cross-talk in ERK MAPK and PI3K/AKT/mTOR pathways68. These pathways co-regulate cell’s growth, differentiation and survival mechanism by feed back loops and cross-activating and cross-inhibiting each other (RAF phosphorylation by AKT, mTORC-1 phosphorylation by ERK1/2)

Many physiological processes in the cells require the participation of both intra- and extra-mitochondrial enzyme reactions. A link between mitochondria and cytosol is provided by a group of proteins known as the mitochondrial carriers (MCs) family (Arco & Satrustegui, 2005; F. Palmieri, 2004). MCs comprise a family of about 40-50 proteins, depending on the organism, and provide the main communication between mitochondrial matrix and extra-mitochondrial spaces by transporting a wide range of metabolites, nucleotides and cofactors.

The cell’s mitochondrial population is normally highly dynamic and exhibits variable turnover rates. The turnover process is accomplished by an actively regulated transcriptional network for mitochondrial replenishment that is coordinated with the degradation and elimination of senescent and damaged mitochondria by selective mitochondrial autophagy or mitophagy. Although mitochondria are constantly renewed, the ongoing rate of homeostatic QC processes in vivo is fairly low (Miwa et al., 2008) on the order of days, whereas in cells, it is more rapid (Hernandez et al., 2013). Mitochondrial turnover in the rat heart has an estimated half-life of roughly two weeks (Rabinowitz and Zak, 1975). Thus, for a typical cardiomyocyte under basal conditions (~1000 mitochondria), 1.5 mitochondria would be replaced each hour. Moreover, mitochondrial turnover may be regulated by the circadian clock as a number of OXPHOS enzymes show strong diurnal variation in expression. This may be related in part to the period of fasting during sleep; therefore mitochondrial turnover at night may be more active with a few percent of the mitochondrial population replaced each night. Mitochondrial turnover rates also vary with specific metabolic status of tissues, but can be greatly

Dr. Kosmider has publications with Dr. Steven Kelsen, who is a co-investigator on this grant. His clinical and research interests are focused on signaling pathways and the pathophysiology of oxidant stress-induced epithelial injury. He has training in respiratory medicine and physiology and will provide knowledge of the lung diseases. He has also collaborated with Dr. Muniswamy Madesh, a co-investigator in this project, who is an expert in studies of mitochondrial dysfunction and mechanisms of mitochondrial induced apoptosis.

The protein usually targets old, dysfunctional proteins from the cell cycle, which makes sense for controlled cell division. The ubiquitination mechanism was originally obscure, but made more clear in the mid-1980’s by Aaron Ciechhanover, Avram Hershko, and Irwin Rose, which helped them win the Nobel Prize for Chemistry in 2004 (Nobel Prize website). Initially, the process was looked at as proteolytic system utilizing ATP. Many ATP-dependent proteolysis factor 1 (APF-1) molecules linked with a substrates that were later found to be deteriorated with free APF-1 in the cellular extract (Ciechhanover, 2012). APF-1 was called ubiquitin and it uses its C-terminal glycine to conjugate to the substrate’s

Mitochondria provide the energy used within our cell to carry the vast variety of actives that is fundamental life (). Without the huge input of ATP derived from the mitochondria, the

Insulin also binds to IGF-1 receptors with low affinity (Smith, 1988) and stimulates the formation of an active Ras-GTP complex and ERK-MAPK pathway (Porras, 1996) and the PI3K pathway. Both these pathways appear to be required for adipogenesis (Sale, 1995; Sakaue, 1998; Tang, 2005). The Ras-GTP complex activates RAF kinase which is phosphorylated to induce MAPK. The phosphorylated MAPK interacts with Elk1. Phohosphorylation of Ekl1 facilitates binding to the mediator complex via the Med23 subunit (Stevens, 2002; Wang, 2005). The mediator complex is a multi-protein complex which bridges gene specific transcription factors and transcriptional machinery. The Mediator Med23 (Sur2) subunit was originally identified as a genetic suppressor of a hyperactive Ras phenotype in C.elegans (Singh, 1995). Elk1 is a member of the ternary complex factor family and an important regulator of adipogenesis. The Elk1/Med23 interaction controls the expression of Krox20 in a MAPK dependent manner by promoting a productive preinitiation complex which leads to transcription (Wang, 2009). Krox20 is expressed in adipose tissue (Soukas, 2001) and is the first transcriptional factor expressed during adipogenesis after insulin stimulation (Chen, 2005; Gonzalez, 2005). Krox20 expression has been reported to regulate