Interaction between ATM Kinase and p53 in determining glioma radiosensitivity

Glioblastoma multiforme (GBM) is the most common primary brain tumor. Studies have shown that targeting the DNA damage response can sensitize cancer cells to DNA damaging agents. Ataxia telangiectasia mutated (ATM) is involved in signaling DNA double strand breaks. Our group has previously shown that ATM inhibitors (ATMi) sensitize GBM cells and tumors to ionizing radiation. This effect is greater when the tumor suppressor p53 is mutated. The goals of this work include validation of a new ATM inhibitor, AZ32, and elucidation of how ATMi and p53 status interact to promote cell death after radiation. We propose that ATMi and radiation induce mitotic catastrophe in p53 mutants by overriding cell cycle arrest. We tested this hypothesis in human colon carcinoma and glioma cells that differ only in p53 status. We found that AZ32 effectively inhibits phosphorylation of ATM targets. In addition, AZ32 significantly sensitizes glioma cells to ionizing radiation. While HCT116 colon carcinoma cells fail to arrest the cell cycle after radiation, their response to ATMi differs from that in gliomas. Indeed, wild type HCT116 cells were more sensitive than p53 mutants to ionizing radiation in the presence of ATMi. In contrast, ATMi significantly radiosensitized glioma cells in which p53 is knocked down. Live cell imaging confirmed that radiation and ATMi preferentially induce mitotic catastrophe in p53-deficient cells. We conclude that p53-deficient cells rely on ATM signaling for G2/M cell cycle arrest. We propose a model of G2/M arrest whereby ATM and p53-dependent signaling pathways converge to ultimately inhibit Cdc25 phosphatases

Citrate Binding to the Membrane Protein Proteorhodopsin

Proteorhodopsin (pR) is an intrinsic membrane protein with an important role in solar-energy storage of the biosphere. Earlier work in our lab has shown that polyhistidine-tagged pR can be purified by means of selective precipitation with citrate under specific conditions, as can a number of mutants based on this His6-tagged pR. Purification of a heterologously-expressed trans-membrane protein by a simple salt such as citrate is novel. However, such a phenomenon leads to several questions: How does citrate cause pR precipitation? Does the polyhistidine-tag assist in such a precipitation? Is this precipitation pH-specific? Does citrate affect the function of pR? Does citrate-induced pR precipitation have any biological significance? Are there other ions that could cause pR precipitation? This dissertation focuses on understanding the nature of the interaction of pR with citrate and other anions, and in particular on trying to take advantage of this interaction in order to develop a novel membrane protein purification method. The end goal that branches out of these two aims is to utilize the compact citrate interaction site identified in pR, by incorporating it into other membrane proteins and using it to permit their purification by similar simple procedures. In Chapter 1, I briefly provide some background information on the wide variety of structurally-similar proteins as rhodopsins that include pR. I also describe the general importance of developing purification methods for 7-helix membrane proteins, including pR. Chapter 2 focuses on the investigation of the nature of citrate-binding site of pR. To address the main question of how citrate aids in pR purification, site-directed mutagenesis technique was applied to generate several single, double, triple or quadruple mutants of pR in a histidine-tag free background, which were then tested for their reactivity to citrate. Several different anions were tested to examine if precipitation of pR was specific to citrate or whether the precipitation is susceptible to other negatively charged salts. Photocycle of pR progresses through several intermediates, each with a distinct absorption maximum (described in subsection 1.3.1). M-intermediate is detected at pH ≥ 8 with λmax = 410 nm. Flash spectroscopy involves excitation of pR at a particular wavelength that leads to transient absorption, thus, signaling the formation of the corresponding intermediate. Flash-induced transient visible absorption measurements were used to assay the effect of exposing pR to citrate on its physiological function. Chapter 3 describes the development of a method of purification of pR using simple salts, citrate and phosphate. Chapter 4 begins an exploration of a future direction. The ultimate objective is to apply the above techniques for the general purpose of 7-helix membrane protein purification, especially for the important class of pharmacological receptors known as GPCRs. An attempt at heterologous expression in E. coli, and purification, of a mammalian GPCR, is described therein. Such a method would be desirable for obtaining proteins for structural, functional and pharmacological studies

Molecular Aspects of Dopaminergic Neurodegeneration: Gene-Environment Interaction in Parkin Dysfunction

Parkinson’s disease (PD) is a common neurodegenerative movement disorder that is characterized pathologically by a progressive loss of midbrain dopaminergic neurons and by protein inclusions, designated Lewy bodies and Lewy neurites. PD is one of the most common neurodegenerative diseases, affecting almost 1% of the population over 60 years old. Although the symptoms and neuropathology of PD have been well characterized, the underlying mechanisms and causes of the disease are still not clear. Genetic mutations can provide important clues to disease mechanism, but most PD cases are sporadic rather than familial; environmental factors have long been suspected to contribute to the disease. Although more than 90% of PD cases occur sporadically and are thought to be due, in part, to oxidative stress and mitochondrial dysfunction, the study of genetic mutations has provided great insight into the molecular mechanisms of PD. Furthermore, rotenone, a widely used pesticide, and paraquat and maneb cause a syndrome in rats and mice that mimics, both behaviorally and neurologically, the symptoms of PD. In the current review, we will discuss various aspects of gene-environment interaction that lead to progressive dopaminergic neurodegenration, mainly focusing on our current finding based on stress-mediated parkin dysfunction

MPEG-4 video transmission using distributed TDMA MAC protocol over IEEE 802.15.4 wireless technology

The issues of green technology nowadays give an inspiration to the researcher to make all the future design to be energy efficient. Medium Access Control (MAC) layer is the most effective layer to provide energy efficient due to its ability to control the physical radio directly. One of the important applications in the future is a video transmission that can be transmitted with low-cost and low power consumption. MPEG-4 is one of the international standards for moving video. MPEG-4 provide better compression and primarily design at low bit rate communication. In order to achieve good quality for video application, the design at MAC layer must be strong. Therefore, to increase the performance of the MPEG-4 in IEEE 802.15.4, in this paper we propose a cross layer design between MAC layer and Application layer. A priority queue will be implemented at MAC scheduling depends on the level of frame important in MPEG-4 format frame. A distributed Time division Multiple Access (TDMA) will be used for MAC protocol to provide reliable data transmission for high priority frame