Medical Pathology Commons^™

Intra-Tumor Heterogeneity Of Mlh1 Promoter Methylation Revealed By Deep Single Molecule Bisulfite Sequencing., Katherine E Varley, David G Mutch, Tina B Edmonston, Paul J Goodfellow, Robi D Mitra

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

A single tumor may contain cells with different somatic mutations. By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis. In contrast, the extent of epigenetic intra-tumor heterogeneity and how it influences tumor biology is under-explored. We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing. We used deep single molecule bisulfite sequencing and sample-specific DNA barcodes to determine the spectrum of MLH1 promoter methylation across an average of 1000 molecules in each of 33 individual samples in parallel, including endometrial cancer, matched blood and normal endometrium. This first glimpse, …

Decorin Is A Novel Antagonistic Ligand Of The Met Receptor., Silvia Goldoni, Ashley Humphries, Alexander Nyström, Sampurna Sattar, Rick T Owens, David J Mcquillan, Keith Ireton, Renato V Iozzo

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Decorin, a member of the small leucine-rich proteoglycan gene family, impedes tumor cell growth by down-regulating the epidermal growth factor receptor. Decorin has a complex binding repertoire, thus, we predicted that decorin would modulate the bioactivity of other tyrosine kinase receptors. We discovered that decorin binds directly and with high affinity (K(d) = approximately 1.5 nM) to Met, the receptor for hepatocyte growth factor (HGF). Binding of decorin to Met is efficiently displaced by HGF and less efficiently by internalin B, a bacterial Met ligand. Interaction of decorin with Met induces transient receptor activation, recruitment of the E3 ubiquitin ligase …

Developing Tta Transgenic Rats For Inducible And Reversible Gene Expression., Hongxia Zhou, Cao Huang, Min Yang, Carlisle P Landel, Pedro Yuxing Xia, Yong-Jian Liu, Xu Gang Xia

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout …

Graft-Versus-Host Disease-Like Pattern In Mycophenolate Mofetil Related Colon Mucosal Injury: Role Of Fish In Establishing The Diagnosis., Kathryn C Behling, Dorothy M J Foster, Tina B Edmonston, Agnieszka K Witkiewicz

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Mycophenolate mofetil (CellCept®), a commonly used immunosuppressive drug in solid organ transplantation, has recently been shown to cause graft-versus-host disease (GVHD)-like changes in the gastrointestinal tract. On rare occasions, true GVHD has also been documented in the gastrointestinal tract of solid organ transplant patients. Because the treatment for these two entities is different, i.e. removal of the offending agent versus the administration of steroids, proper identification of the cause is imperative. We present a case of mycophenolate mofetil colitis mimicking grade I GVHD of the gut. In our study, we used fluorescence in situ hybridization for the Y chromosome to …

A New Model For Hemoglobin Ingestion And Transport By The Human Malaria Parasite Plasmodium Falciparum., Michelle D Lazarus, Timothy G Schneider, Theodore F Taraschi

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The current model for hemoglobin ingestion and transport by intraerythrocytic Plasmodium falciparum malaria parasites shares similarities with endocytosis. However, the model is largely hypothetical, and the mechanisms responsible for the ingestion and transport of host cell hemoglobin to the lysosome-like food vacuole (FV) of the parasite are poorly understood. Because actin dynamics play key roles in vesicle formation and transport in endocytosis, we used the actin-perturbing agents jasplakinolide and cytochalasin D to investigate the role of parasite actin in hemoglobin ingestion and transport to the FV. In addition, we tested the current hemoglobin trafficking model through extensive analysis of serial …

Gating Charge Immobilization In Kv4.2 Channels: The Basis Of Closed-State Inactivation., Kevin Dougherty, Jose A De Santiago-Castillo, Manuel Covarrubias

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Kv4 channels mediate the somatodendritic A-type K+ current (I(SA)) in neurons. The availability of functional Kv4 channels is dynamically regulated by the membrane potential such that subthreshold depolarizations render Kv4 channels unavailable. The underlying process involves inactivation from closed states along the main activation pathway. Although classical inactivation mechanisms such as N- and P/C-type inactivation have been excluded, a clear understanding of closed-state inactivation in Kv4 channels has remained elusive. This is in part due to the lack of crucial information about the interactions between gating charge (Q) movement, activation, and inactivation. To overcome this limitation, we engineered a charybdotoxin …

Silencing Of The Pink1 Gene Expression By Conditional Rnai Does Not Induce Dopaminergic Neuron Death In Mice., Hongxia Zhou, Björn H Falkenburger, Jörg B Schulz, Kim Tieu, Zuoshang Xu, Xu Gang Xia

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA …

No Vaccine Against Hiv Yet--Are We Not Perfectly Equipped?, Mahender Singh

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Enormous effort has been devoted to the development of a vaccine against human immunodeficiency virus (HIV). But it is proving to be an unprecedented challenge to create an effective vaccine mainly due to the high genetic variability of the virus and the necessity of cytotoxic T lymphocytes (CTL) for containing the infection. Currently pursued vaccine strategies appear to induce CTL in nonhuman primate models but in the early clinical trials, these strategies fail to fully control the viral infection. New strategies that can cover the vast genetic diversity of HIV are needed for the development of a potent vaccine.

Selective Role For Superoxide In Insp3 Receptor-Mediated Mitochondrial Dysfunction And Endothelial Apoptosis., Muniswamy Madesh, Brian J Hawkins, Tatyana Milovanova, Cunnigaiper D Bhanumathy, Suresh K Joseph, Satish P Ramachandrarao, Kumar Sharma, Tomohiro Kurosaki, Aron B Fisher

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane …

Functionally Active T1-T1 Interfaces Revealed By The Accessibility Of Intracellular Thiolate Groups In Kv4 Channels., Guangyu Wang, Mohammad Shahidullah, Carmen A Rocha, Candace Strang, Paul J Pfaffinger, Manuel Covarrubias

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is …

Control Of Mitochondrial Motility And Distribution By The Calcium Signal: A Homeostatic Circuit., Muqing Yi, David Weaver, György Hajnóczky

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Mitochondria are dynamic organelles in cells. The control of mitochondrial motility by signaling mechanisms and the significance of rapid changes in motility remains elusive. In cardiac myoblasts, mitochondria were observed close to the microtubular array and displayed both short- and long-range movements along microtubules. By clamping cytoplasmic [Ca2+] ([Ca2+]c) at various levels, mitochondrial motility was found to be regulated by Ca2+ in the physiological range. Maximal movement was obtained at resting [Ca2+]c with complete arrest at 1-2 microM. Movement was fully recovered by returning to resting [Ca2+]c, and inhibition could be repeated with no apparent desensitization. The inositol 1,4,5-trisphosphate- or …

Signaling Switches And Bistability Arising From Multisite Phosphorylation In Protein Kinase Cascades., Nick I Markevich, Jan B. Hoek, Boris N. Kholodenko

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Mitogen-activated protein kinase (MAPK) cascades can operate as bistable switches residing in either of two different stable states. MAPK cascades are often embedded in positive feedback loops, which are considered to be a prerequisite for bistable behavior. Here we demonstrate that in the absence of any imposed feedback regulation, bistability and hysteresis can arise solely from a distributive kinetic mechanism of the two-site MAPK phosphorylation and dephosphorylation. Importantly, the reported kinetic properties of the kinase (MEK) and phosphatase (MKP3) of extracellular signal-regulated kinase (ERK) fulfill the essential requirements for generating a bistable switch at a single MAPK cascade level. Likewise, …

Stretch-Induced Calcium Release In Smooth Muscle., Guangju Ji, Robert J Barsotti, Morris E Feldman, Michael I Kotlikoff

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) …

Vdac-Dependent Permeabilization Of The Outer Mitochondrial Membrane By Superoxide Induces Rapid And Massive Cytochrome C Release., M Madesh, György Hajnóczky

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Enhanced formation of reactive oxygen species (ROS), superoxide (O2*-), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2*- elicited rapid and massive cytochrome c release (CCR), whereas H2O2 failed to induce any release. Both O2*- and H2O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2*--induced CCR. Furthermore, O2*- alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential …

Eliciting The Low-Activity Aldehyde Dehydrogenase Asian Phenotype By An Antisense Mechanism Results In An Aversion To Ethanol., E Garver, Tu Gc, Q N Cao, M Aini, F Zhou, Y Israel

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2-2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2-2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 …

Acidification-Induced Sensitization To Thermoradiotherapy In Breast Cancer, Dennis B. Leeper, L T. Komarnicky

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Hyperthermia is an extensively studied cytotoxic agent, with strong radio- and chemosensitizing potential. Recent positive clinical trials combining superficial or deep heating techniques with radiation therapy strongly support a role for hyperthermia as an adjuvant to radiation. Many in vitro and in vivo studies have shown that acute extracellular acidification will compromise fundamental protective cellular responses and enhance tumor response to hyperthermia and chemotherapy.

Breast cancers, like most other tumors, exhibit elevated levels of lactate production that provides a basis for selective acidification. A phase I/II clinical trial is underway to test the hypothesis that hyperglycemia-induced acute acidification will sensitize …

Single-Channel Properties In Endoplasmic Reticulum Membrane Of Recombinant Type 3 Inositol Trisphosphate Receptor., D O Mak, S Mcbride, V Raghuram, Y Yue, Suresh K. Joseph, J K Foskett

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel …