Medical Molecular Biology Commons^™

The Biochemical Pathway Leading To Lpa Generation Upon Blood Coagulation, Alyssa Lynn Jefferson Bolen

Theses and Dissertations (ETD)

Platelet activation initiates an upsurge in 18:2 and 20:4 lysophosphatidic acid (LPA) production. The biochemical pathway responsible for LPA production during blood clotting is not fully understood. We have purified a phospholipase A₁ (PLA₁) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate‑biotin affinity labeling and proteomics. We identified acyl‑protein thioesterase 1 (aka. lysophospholipase A1, accession code O75608) as a novel PLA₁. Addition of this recombinant PLA₁ significantly increased the production of sn‑2‑esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We next examined the regioisomeric preference of lysophospholipase …

Characterization Of The Sigma Factor Proteins And The Dna Binding Protein Euo Of Chlamydia, Cory L. Blackwell

Theses and Dissertations (ETD)

Chlamydia spp. are prokaryotic obligate intracellular pathogens with a unique, biphasic developmental cycle in which an infectious, extracellular form termed the elementary body (EB) interconverts with a metabolically active intracellular reticulate body (RB) within host cells. Subsets of genes are differentially expressed during the developmental cycle, and these genes are believed to be responsible for the transitions between the EB and RB forms. The goal of these studies was to explore two potential mechanisms that may function in regulating developmental cycle stage‑specific gene expression in chlamydiae: a cascade of sigma factor expression and the binding of the early stage protein …

Muc4 Stabilizes Her2 Expression And Maintains The Cancer Stem Cell Population In Ovarian Cancer Cells., Moorthy P. Ponnusamy, Parthasarathy Seshacharyulu, Arokia P. Vaz, Parama Dey, Surinder K. Batra

Journal Articles: Biochemistry & Molecular Biology

BACKGROUND: Recent evidence has suggested that the capability of cancer to grow, propagate and relapse after therapy is dependent on a small subset of the cell population within the tumor, called cancer stem cells. Therefore, this subpopulation of cells needs to be targeted with different approaches by identification of unique stem-cell specific target antigens. One of the well known tumor antigens is the epithelial cell mucin MUC4, which is aberrantly expressed in ovarian cancer as compared to the normal ovary and plays a pivotal role in the aggressiveness and metastasis of ovarian cancer cells. In the present study, we aimed …

Human Papillomavirus Genotype Concordance Within Zambian Couples, Kgomotso Makhaola

School of Biological Sciences: Dissertations, Theses, and Student Research

Human Papillomavirus (HPV) is strongly associated with the development of cervical cancer, and has also been identified in other anogenital cancers such as penile, oral, and anal cancers. In regions like Sub Saharan Africa where the prevalence of HIV/AIDS is high,   increased rates of HPV infections have also been observed, however the effect of HIV/AIDS on the transmission of HPV is not yet well understood.  In this study specimens for HIV, HPV testing and pathology were collected from male participants and their female partners at a Urology department in Lusaka University Teaching Hospital, Zambia. Seventy four samples were collected but …

Activated Krasg¹²D Is Associated With Invasion And Metastasis Of Pancreatic Cancer Cells Through Inhibition Of E-Cadherin., Satyanarayana Rachagani, S Senapati, S Chakraborty, Moorthy P. Ponnusamy, Sushil Kumar, Lynette Smith, Maneesh Jain, Surinder K. Batra

Journal Articles: Biochemistry & Molecular Biology

BACKGROUND: Pancreatic cancer (PC) harbours an activated point mutation (Kras(G12D)) in the Kras proto-oncogene that has been demonstrated to promote the development of PC.

METHODS: This study was designed to investigate the effect of the oncogenic Kras(G12D) allele on aggressiveness and metastatic potential of PC cells. We silenced the oncogenic Kras(G12D) allele expression in CD18/HPAF and ASPC1 cell lines by stable expression of shRNA specific to the Kras(G12D)allele.

RESULTS: The Kras(G12D) knockdown cells exhibited a significant decrease in motility (P

CONCLUSIONS: The results of this study suggest that the Kras(G12D) allele promotes metastasis in PC cells partly through the downregulation …

Progressive Multifocal Leukoencephalopathy In A Hiv-Negative Patient With Small Lymphocytic Leukemia Following Treatment With Rituximab., Subhankar Chakraborty, Stefano R. Tarantolo, John Treves, David Sambol, Ralph J. Hauke, Surinder K. Batra

Journal Articles: Biochemistry & Molecular Biology

We describe a case of progressive multifocal leukoencephalopathy (PML) caused by infection with the human polyomavirus JC virus in a patient with B-cell small lymphocytic leukemia who was treated with rituximab. The first symptoms of PML appeared immediately following the last of five cycles of rituximab, cyclophosphamide and pentostatin. Magnetic resonance imaging revealed changes consistent with PML, although JC virus DNA was not detected by polymerase chain reaction assay of the cerebrospinal fluid. A stereotactic biopsy of the brain showed histological changes consistent with PML, while electron microscopy revealed JC virus particles attached to the nuclei of astrocytes. The patient …

Hedgehog Inhibition Promotes A Switch From Type Ii To Type I Cell Death Receptor Signaling In Cancer Cells., Satoshi Kurita, Justin L. Mott, Sophie C. Cazanave, Christian D. Fingas, Maria E. Guicciardi, Steve F. Bronk, Lewis R. Roberts, Martin E. Fernandez-Zapico, Gregory J. Gores

Journal Articles: Biochemistry & Molecular Biology

TRAIL is a promising therapeutic agent for human malignancies. TRAIL often requires mitochondrial dysfunction, referred to as the Type II death receptor pathway, to promote cytotoxicity. However, numerous malignant cells are TRAIL resistant due to inhibition of this mitochondrial pathway. Using cholangiocarcinoma cells as a model of TRAIL resistance, we found that Hedgehog signaling blockade sensitized these cancer cells to TRAIL cytotoxicity independent of mitochondrial dysfunction, referred to as Type I death receptor signaling. This switch in TRAIL requirement from Type II to Type I death receptor signaling was demonstrated by the lack of functional dependence on Bid/Bim and Bax/Bak, …

Transcriptional Profiling Of Peripheral Blood Mononuclear Cells In Pancreatic Cancer Patients Identifies Novel Genes With Potential Diagnostic Utility., Michael J. Baine, Subhankar Chakraborty, Lynette M. Smith, Kavita Mallya, Aaron R. Sasson, Randall E. Brand, Surinder K. Batra

Journal Articles: Biochemistry & Molecular Biology

BACKGROUND: It is well known that many malignancies, including pancreatic cancer (PC), possess the ability to evade the immune system by indirectly downregulating the mononuclear cell machinery necessary to launch an effective immune response. This knowledge, in conjunction with the fact that the trancriptome of peripheral blood mononuclear cells has been shown to be altered in the context of many diseases, including renal cell carcinoma, lead us to study if any such alteration in gene expression exists in PC as it may have diagnostic utility.

METHODS AND FINDINGS: PBMC samples from 26 PC patients and 33 matched healthy controls were …

Three Dimensional Projection Environment For Molecular Design And Surgical Simulation, Matthew Wampole, Eric Wickstrom, Chang-Po Chen, Devakumar Devadhas, Yuan-Yuan Jin, Jeffrey M. Sanders, John C. Kairys, Martha L. Ankeny, Rui Hu, Kenneth E. Barner, Karl V. Steiner, Mathew L. Thakur

Department of Biochemistry and Molecular Biology Faculty Papers

Poster presented at "Medicine Meets Virtual Reality" conference February 8-12, 2011 in Newport Beach, California.

Conclusions:

Turning 2D CT/PET slices into 3D objects assists in understanding the topology surrounding tumor masses. Incorporating the visual and physical characteristics of a patient’s anatomy will provide surgeons with an informative pre-operative tool to plan and practice the operation before the first incision. Including haptic feedback provides a familiar 'feel' to surgeons as they palpate the target organ, trying to locate the tumor and determine how large a margin of resection will be needed. The development of genetic PET imaging and contrast CT into …

Steroids Up-Regulate P66shc Longevity Protein In Growth Regulation By Inhibiting Its Ubiquitination., Santosh Kumar, Satyendra Kumar, Mythilypriya Rajendran, Syed Mahfuzul Alam, Fen-Fen Lin, Pi-Wan Cheng, Ming-Fong Lin

Journal Articles: Biochemistry & Molecular Biology

BACKGROUND: p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism.

METHODS AND FINDINGS: In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on …

Cellular Inhibitor Of Apoptosis 1 (Ciap-1) Degradation By Caspase 8 During Tnf-Related Apoptosis-Inducing Ligand (Trail)-Induced Apoptosis., Maria Eugenia Guicciardi, Justin L. Mott, Steven F. Bronk, Satoshi Kurita, Christian D. Fingas, Gregory J. Gores

Journal Articles: Biochemistry & Molecular Biology

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as …

Mir-27b*, An Oxidative Stress-Responsive Microrna Modulates Nuclear Factor-Kb Pathway In Raw 264.7 Cells, Sivasubramani Thulasingam, Chandirasegaran Massilamany, Arunakumar Gangaplara, Hongjiu Dai, Shahlo Yarbaeva, Sakthivel Subramaniam, Jean-Jack Riethoven, James Eudy, Marjorie F. Lou, Jay Reddy

Jay Reddy Publications

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H₂O₂) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H₂O₂. These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, …

Identification Of A Second Mimicry Epitope From Acanthamoeba Castellanii That Induces Cns Autoimmunity By Generating Cross-Reactive T Cells For Mbp 89–101 In Sjl Mice, Chandirasegaran Massilamany, Oluwatoyin A. Asojo, Arunakumar Gangaplara, David J. Steffen, Jay Reddy

Jay Reddy Publications

We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139–151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89–101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108–120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89–101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108–120 develop encephalomyelitis similar to the disease induced by the cognate peptide. …

Identification Of A Second Mimicry Epitope From Acanthamoeba Castellanii That Induces Cns Autoimmunity By Generating Cross-Reactive T Cells For Mbp 89–101 In Sjl Mice, Chandirasegaran Massilamany, Oluwatoyin A. Asojo, Arunakumar Gangaplara, David J. Steffen, Jay Reddy

Jay Reddy Publications

We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139–151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89–101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108–120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89–101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108–120 develop encephalomyelitis similar to the disease induced by the cognate peptide. …

The Role Of The Nr4a Orphan Nuclear Receptor Nor1 In Vascular Cells And Atherosclerosis, Yue Zhao

University of Kentucky Doctoral Dissertations

The neuron-derived orphan receptor 1 (NOR1) belongs to the NR4A nuclear receptor subfamily. As an immediate early response gene, NOR1 is rapidly induced by a broad spectrum of physiological and pathological signals. Functional studies demonstrate NOR1 as a constitutively active ligand-independent nuclear receptor whose transcriptional activity is dependent on both expression level and posttranslational modifications. To date, an increasing number of studies have demonstrated a pivotal role of NOR1 in the transcriptional control of metabolism and the development of cardiovascular diseases.

In this dissertation, we demonstrate NOR1 expression in endothelial cells and sub-endothelial cells of human atherosclerotic lesions. In response …

Protein Modification By Arginylation, Hideko Kaji, Akira Kaji

Department of Biochemistry and Molecular Biology Faculty Papers

The modification of protein by arginine catalyzed by arginyltransferases (ATE1) described by the Kashina group in this issue shows that arginylation of protein occurs widely in biology and is being recognized as a key regulatory reaction such as phosphorylation of proteins (Wang et al., 2011).

Carhsp1 Is Required For Effective Tumor Necrosis Factor Alpha Mrna Stabilization And Localizes To Processing Bodies And Exosomes, Jason R. Pfeiffer, Bethany L. Mcavoy, Ryan E. Fecteau, Kristen M. Deleault, Seth A. Brooks

Dartmouth Scholarship

Tumor necrosis factor alpha (TNF-α) is a critical mediator of inflammation, and its production is tightly regulated, with control points operating at nearly every step of its biosynthesis. We sought to identify uncharacterized TNF-α 3' untranslated region (3'UTR)-interacting proteins utilizing a novel screen, termed the RNA capture assay. We identified CARHSP1, a cold-shock domain-containing protein. Knockdown of CARHSP1 inhibits TNF-α protein production in lipopolysaccharide (LPS)-stimulated cells and reduces the level of TNF-α mRNA in both resting and LPS-stimulated cells. mRNA stability assays demonstrate that CARHSP1 knockdown decreases TNF-α mRNA stability from a half-life (t(1/2)) of 49 min to a t(1/2) …

A Smac Mimetic Reduces Tnf Related Apoptosis Inducing Ligand (Trail)-Induced Invasion And Metastasis Of Cholangiocarcinoma Cells., Christian D. Fingas, Boris R.A. Blechacz, Rory L. Smoot, Maria E. Guicciardi, Justin L. Mott, Steve F. Bronk, Nathan W. Werneburg, Alphonse E. Sirica, Gregory J. Gores

Journal Articles: Biochemistry & Molecular Biology

UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand that, failing to kill CCA cells, instead promotes their tumorigenicity and especially the metastatic behaviors of cell migration and invasion. Second mitochondria-derived activator of caspase (smac) mimetics are promising cancer therapeutic agents that enhance proapoptotic death receptor signaling by causing cellular degradation of inhibitor of apoptosis (IAP) proteins. Our aim was to examine the in vitro and in vivo effects of the smac mimetic JP1584 in CCA. Despite JP1584-mediated loss of cellular inhibitor of apoptosis-1 (cIAP-1) and cIAP-2, TRAIL failed to induce apoptosis in KMCH-1, …

Transcriptional Suppression Of Mir-29b-1/Mir-29a Promoter By C-Myc, Hedgehog, And Nf-Kappab., Justin L. Mott, Satoshi Kurita, Sophie C. Cazanave, Steven F. Bronk, Nathan W. Werneburg, Martin E. Fernandez-Zapico

Journal Articles: Biochemistry & Molecular Biology

MicroRNAs regulate pathways contributing to oncogenesis, and thus the mechanisms causing dysregulation of microRNA expression in cancer are of significant interest. Mature mir-29b levels are decreased in malignant cells, and this alteration promotes the malignant phenotype, including apoptosis resistance. However, the mechanism responsible for mir-29b suppression is unknown. Here, we examined mir-29 expression from chromosome 7q32 using cholangiocarcinoma cells as a model for mir-29b downregulation. Using 5' rapid amplification of cDNA ends, the transcriptional start site was identified for this microRNA locus. Computational analysis revealed the presence of two putative E-box (Myc-binding) sites, a Gli-binding site, and four NF-kappaB-binding sites …

Noxa Mediates Hepatic Stellate Cell Apoptosis By Proteasome Inhibition., Ivette M. Sosa Seda, Justin L. Mott, Yuko Akazawa, Fernando J. Barreyro, Steven F. Bronk, Scott H. Kaufmann, Gregory J. Gores

Journal Articles: Biochemistry & Molecular Biology

Aim: Induction of hepatic stellate cell (HSC) apoptosis is a viable therapeutic strategy to reduce liver fibrogenesis. Although BH3-only proteins of the Bcl-2 family trigger pro-apoptotic pathways, the BH3-only proteins mediating HSC apoptosis have not been well defined. Our aim, using proteasome inhibition as a model to induce HSC apoptosis, was to examine the BH3-only proteins contributing to cell death of this key liver cell subtype. Methods: Apoptosis was induced by treating LX-2 cells, an immortalized human hepatic stellate cell line, and primary rat stellate cells with the proteasome inhibitor MG-132. Results: Treatment with proteasome inhibitors increased expression of Noxa …