Medicine and Health Sciences Commons^™

Dancing Through Life: Molecular Dynamics Simulations And Network-Centric Modeling Of Allosteric Mechanisms In Hsp70 And Hsp110 Chaperone Proteins, Gabrielle Stetz, Gennady M. Verkhivker

Mathematics, Physics, and Computer Science Faculty Articles and Research

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that …

Chemical-Free Technique To Study The Ultrastructure Of Primary Cilium, Ashraf M. Mohieldin, Wissam A. Aboualaiwi, Min Gao, Surya M. Nauli

Pharmacy Faculty Articles and Research

A primary cilium is a hair-like structure with a width of approximately 200 nm. Over the past few decades, the main challenge in the study of the ultrastructure of cilia has been the high sensitivity of cilia to chemical fixation, which is required for many imaging techniques. In this report, we demonstrate a combined high-pressure freezing (HPF) and freeze-fracture transmission electron microscopy (FFTEM) technique to examine the ultrastructure of a cilium. Our objective is to develop an optimal high-resolution imaging approach that preserves cilia structures in their best natural form without alteration of cilia morphology by chemical fixation interference. Our …

Real-Time Detection Of Breast Cancer Cells Using Peptidefunctionalized Microcantilever Arrays, Hashem Etayash, Keren Jiang, Sarfuddin Azmi, Thomas Thundat, Kamaljit Kaur

Pharmacy Faculty Articles and Research

Ligand-directed targeting and capturing of cancer cells is a new approach for detecting circulating tumor cells (CTCs). Ligands such as antibodies have been successfully used for capturing cancer cells and an antibody based system (CellSearch®) is currently used clinically to enumerate CTCs. Here we report the use of a peptide moiety in conjunction with a microcantilever array system to selectively detect CTCs resulting from cancer, specifically breast cancer. A sensing microcantilever, functionalized with a breast cancer specific peptide 18-4 (WxEAAYQrFL), showed significant deflection on cancer cell (MCF7 and MDA-MB-231) binding compared to when exposed to noncancerous (MCF10A and HUVEC) cells. …

Elongation Factor-P At The Crossroads Of The Host-Endosymbiont Interface, Andrei Rajkovic, Anne Witzky, William Navarre, Andrew J. Darwin, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is an ancient bacterial translational factor that aids the ribosome in polymerizing oligo-prolines. EF-P structurally resembles tRNA and binds in-between the exit and peptidyl sites of the ribosome to accelerate the intrinsically slow reaction of peptidyl-prolyl bond formation. Recent studies have identified in separate organisms, two evolutionarily convergent EF-P post-translational modification systems (EPMS), split predominantly between gammaproteobacteria, and betaproteobacteria. In both cases EF-P receives a post-translational modification, critical for its function, on a highly conserved residue that protrudes into the peptidyl-transfer center of the ribosome. EPMSs are comprised of a gene(s) that synthesizes the precursor molecule …

Negative Skeletal Effects Of Locally Produced Adiponectin, Marcia J. Abbott, Theresa M. Roth, Linh Ho, Liping Wang, Dylan O'Carroll, Robert A. Nissenson

Health Sciences and Kinesiology Faculty Articles

Epidemiological studies show that high circulating levels of adiponectin are associated with low bone mineral density. The effect of adiponectin on skeletal homeostasis, on osteoblasts in particular, remains controversial. We investigated this issue using mice with adipocyte-specific over-expression of adiponectin (AdTg). MicroCT and histomorphometric analysis revealed decreases (15%) in fractional bone volume in AdTg mice at the proximal tibia with no changes at the distal femur. Cortical bone thickness at mid-shafts of the tibia and at the tibiofibular junction was reduced (3–4%) in AdTg mice. Dynamic histomorphometry at the proximal tibia in AdTg mice revealed inhibition of bone formation. AdTg …

Effect Of Sirna Pre-Exposure On Subsequent Response To Sirna Therapy, Hamidreza Montazeri Aliabadi, Parvin Mahdipoor, Cezary Kucharski, Nicole Chan, Hasan Uludag

Pharmacy Faculty Articles and Research

PURPOSE. An alternative cancer therapy based on RNA interference (RNAi) has shown considerable promise but the possibility of resistance development is not known. This study explored the possibility of therapeutic resistance against siRNA nanoparticles in human cancer cells. METHODS. Two approaches to siRNA treatment were undertaken using lipid-modified polyethylenimines, a single high concentration (shock) and repeated increasing concentrations (gradual). The targets were Mcl-1, RPS6KA5 and KSP in MDA-MB-435 cells. RESULTS. There was no evidence of resistance development in shock-treated cells, while the decrease in mRNA levels of targeted proteins was not as robust in naïve cells in gradual treatment. However, …

Cationic Cell-Penetrating Peptides Are Potent Furin Inhibitors, Bruno Ramos-Molina, Adam N. Lick, Amir Nasrolahi Shirazi, Donghoon Oh, Rakesh Tiwari, Naglaa Salem El-Sayed, Keykavous Parang, Iris Lindberg

Pharmacy Faculty Articles and Research

Cationic cell-penetrating peptides have been widely used to enhance the intracellular delivery of various types of cargoes, such as drugs and proteins. These reagents are chemically similar to the multi-basic peptides that are known to be potent proprotein convertase inhibitors. Here, we report that both HIV-1 TAT47-57 peptide and the Chariot reagent are micromolar inhibitors of furin activity in vitro. In agreement, HIV-1 TAT47-57 reduced HT1080 cell migration, thought to be mediated by proprotein convertases, by 25%. In addition, cyclic polyarginine peptides containing hydrophobic moieties which have been previously used as transfection reagents also exhibited potent furin inhibition in vitro …

Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance In Pseudomonas Aeruginosa, Andrei Rajkovic, Sarah Erickson, Anne Witzky, Owen E. Branson, Jin Seo, Philip R. Gafken, Michael A. Frietas, Julian P. Whitelegge, Kym F. Faull, William Wiley Navarre, Andrew J. Darwin, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which β-Lys is attached to E. coli EF-P. Analysis of the genomes …

Puffs And Tufts: A Comparison Of Trichodesmium Colony Formations And Nutrient Availability Across The North Atlantic Ocean Using Remote Sensing Methods, Marc Rosenfield

Student Scholar Symposium Abstracts and Posters

Trichodesmium, a genus of diazotrophic bacteria, has the capability and the population to produce a large percentage of the total oceanic N2-fixation. Though their population is known to be heavily dependent on two of the ocean’s largest limiting factors, phosphorus and iron concentrations, it is unknown what affect these factors have on the population. In this study two of the largest colony formations of Trichodesmium in the North Atlantic, tufts and puffs, are compared nutrient quality with respect to time and geographical location. Though very little nutrient in situ data was collected from the cruise, remote sensing data collected from …

Effect Of Curcumin Analog Ca27 On Androgen Receptor Translocation In Prostate Cancer Cells, Lijah Vann Gardner

Student Scholar Symposium Abstracts and Posters

The androgen receptor (AR) plays an essential role in promoting the development and progression of metastatic prostate cancer and represents an important molecular target for therapeutic intervention. We have recently described a series of synthetic analogs of the natural product diferuloylmethane (curcumin), some of which induce the down-regulation of AR expression in prostate cancer cells by an as yet largely unknown mechanism of action. While such analogs may in the long term be lead structures for the development of therapeutic drugs, we hypothesize here that they represent ideal molecular probes to identify the mechanism(s) of action for AR down-regulation. We …

Analyzation Of Metabolic Reprogramming In Drug-Resistant Mcf-7 Cells, Derick Han, Ho Leung, Andrew Vo

Student Scholar Symposium Abstracts and Posters

The Warburg effect states that cancer cells mainly receive their energy from anaerobic glycolysis. Thus, mitochondria play a different role in the metabolism of cancer cells as opposed to normal, healthy cells. In chemotherapy, there is always a chance of the cancer regressing. Making drug-resistant cancer cells to analyze their metabolism may change how cancer is treated. This study aimed to create drug-resistant MCF-7 cell lines with doxorubicin in order to determine the metabolic changes that have occurred in the process of becoming resistant to drug treatments.

Effects Of Human Recreational Activity On The Tameness Of Common Loons (Gavia Immer) In Northern Wisconsin, Seth Yund

Student Scholar Symposium Abstracts and Posters

The Common Loon (Gavia immer) is an aquatic diving bird that lives in freshwater habitats in Canada and the northern U.S.. Human activity on a loon’s resident lake may affect its fitness and behavior, yet few studies identify or quantify these effects. We modified existing techniques that measure escape distances in other species to measure tameness as the distance at which individual loons dove in response to human approach by canoe. Tameness was similar between pair members, suggesting that common lake conditions or the behavior of a mate might influence the behavior. Sex, size within sex, and human activity did …

Clinical Applications Of A Combination Chemotherapy Using 8-Chloro Camp And 8-Chloro Adenosine, Erik Munoz, Andrea Saich, Andrew Cox, Yu-An Peter Chang

Student Scholar Symposium Abstracts and Posters

Dr. Cho-Chung from the NIH first thought to use halogenated cAMP derivatives as competitive inhibitors of cAMP to slow down cancer cell mitosis. While the iodine and bromine substituted versions showed very little therapeutic actions, 8-Chloro cAMP has been shown to have strong anti-cancer effects. This has been shown in the phase II clinical trials this drug has undergone. However, these trials have had issues with solubility and toxicity. The drug is similar to vitamin C and is excreted quickly. Scientists tried to overcome this by using a peristaltic pump to give patients a continuous dosage, but this proved too …

Exploring Egr-1 As A Master Regulator Of Prostate Field Cancerization, Kristin Gabriel, Marco Bisoffi

Student Scholar Symposium Abstracts and Posters

Field cancerization denotes the presence of molecular aberrations (genetic, epigenetic, biochemical) in structurally intact cells residing in histologically normal tissues adjacent to tumors. Markers of field cancerization in prostate tissues have the potential to improve the clinical management of this malignancy through their potential to act as indicators of early disease and to serve as molecular targets for early intervention. However, for this, a detailed understanding of the functional pathways underlying field cancerization is necessary. We have recently identified four protein markers of prostate field cancerization, i.e. the key transcription factor early growth response 1 (EGR-1), the lipogenic enzyme fatty …

Prostate Field Cancerization – Thinking Outside The Tumor, Dor Shoshan

Student Scholar Symposium Abstracts and Posters

Analysis of tumor adjacent tissue is assumed to reveal a temporal record of molecular pathways that define oncogenesis. The present study determines expression of the key transcription factor and potential marker of field cancerization early growth response 1 (EGR-1) in human prostate tissues derived from prostatectomies and biopsies. Expression was detected using immunofluorescence and quantified using ImageJ software. Accordingly, EGR-1 expression was similar in cancerous and in histologically normal adjacent tissues from prostatectomy and biopsy specimens. EGR-1 could be exploited as pre-surgical disease indicator in false negative biopsies, identify areas of repeat biopsy, and add molecular information to surgical margins.

Effect Of Novel Dietary Supplement On Metabolism In Vitro And In Vivo, Robert A. Vaughan, Ailish C. White, Jason R. Beam, Nicholas P. Gannon, Randi Garcia-Smith, Roy M. Salgado, Marco Bisoffi, Kristina A. Trujillo, Carole A. Conn, Christine M. Mermier

Health Sciences and Kinesiology Faculty Articles

Obesity is an increasingly prevalent and preventable morbidity with multiple behavioral, surgical and pharmacological interventions currently available. Commercial dietary supplements are often advertised to stimulate metabolism and cause rapid weight and/or fat loss, although few well-controlled studies have demonstrated such effects. We describe a commercially available dietary supplement (purportedly containing caffeine, catechins, and other metabolic stimulators) on resting metabolic rate in humans, and on metabolism, mitochondrial content, and related gene expression in vitro. Human males ingested either a placebo or commercially available supplement (RF) in a randomized double-blind placebo-controlled cross-over fashion. Metabolic rate, respiratory exchange ratio, and blood pressure were …

Live Imaging Of The Ependymal Cilia In The Lateral Ventricles Of The Mouse Brain, Alzahra J. Al Omran, Hannah C. Saternos, Tongyu Liu, Surya M. Nauli, Wissam A. Aboualaiwi

Pharmacy Faculty Articles and Research

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo’s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. …

Effects Of Cannabidiol On Contractions And Calcium Signaling In Rat Ventricular Myocytes, Ramez M. Ali, Lina T. Al Kury, Keun-Hang Susan Yang, Anwar Qureshi, Mohanraj Rajesh, Sehamuddin Galadari, Yaroslav M. Shuba, Frank Christopher Howarth, Murat Oz

Mathematics, Physics, and Computer Science Faculty Articles and Research

Cannabidiol (CBD), a major nonpsychotropic cannabinoid found in Cannabis plant, has been shown to influence cardiovascular functions under various physiological and pathological conditions. In the present study, the effects of CBD on contractility and electrophysiological properties of rat ventricular myocytes were investigated. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the Ca2+ sensitive fluorescent indicator fura-2 AM. Whole-cell patch clamp was used to measure action potential and Ca2+ currents. Radioligand binding was employed to study pharmacological characteristics of CBD binding. CBD (1 μM) caused a significant decrease in the amplitudes of …

Targeting Cell Cycle Proteins In Breast Cancer Cells With Sirna By Using Lipid-Substituted Polyethylenimines, Manoj Parmar, Hamidreza Montazeri Aliabadi, Parvin Mahdipoor, Cezary Kucharski, Robert Maranchuk, Judith C. Hugh, Hasan Uludag

Pharmacy Faculty Articles and Research

The cell cycle proteins are key regulators of cell cycle progression whose de-regulation is one of the causes of breast cancer. RNA interference (RNAi) is an endogenous mechanism to regulate gene expression and it could serve as the basis of regulating aberrant proteins including cell cycle proteins. Since the delivery of small interfering RNA (siRNA) is a main barrier for implementation of RNAi therapy, we explored the potential of a non-viral delivery system, 2.0 kDa polyethylenimines substituted with linoleic acid and caprylic acid, for this purpose. Using a library of siRNAs against cell cycle proteins, we identified cell division cycle …

Transfer Rna Comes Of Age, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

"The year the journal RNA was founded was slated by some in scientific publishing to be the year that one particular type of RNA's run in the spotlight would end. In 1995 I had recently started as a post-doc with Dieter Söll at Yale when he came into the lab to solemnly inform us all that an editor at a certain (S)cience journal had just told him “we won't be publishing any more tRNA papers.” For a post-doc who had migrated across the Atlantic for the sole purpose of furthering his career by working on tRNA this was not great …

Dopaminergic Signaling Within The Primary Cilia In The Renovascular System, Kimberly F. Atkinson, Samred H. Kathem, Xingjian Jin, Brian S. Muntean, Wissam A. Aboualaiwi, Andromeda M. Nauli, Surya M. Nauli

Pharmacy Faculty Articles and Research

Activation of dopamine receptor type-5 (DR5) has been known to reduce systemic blood pressure, most likely by increasing renal vasodilation and enhancing natriuresis in the kidney. However, the mechanism of DR5 in natriuresis and vasodilation was not clearly known. We have previously shown that DR5 is localized to primary cilia of proximal renal epithelial and vascular endothelial cells. We here show that selective activation of DR5 specifically induces calcium influx only in the primary cilia, whereas non-selective activation of dopamine receptor induces calcium fluxes in both cilioplasm and cytoplasm. Cilia-independent signaling induced by thrombin only shows calcium signaling within cytoplasm. …

Kaposi Sarcoma Herpesvirus Induces Ho-1 During De Novo Infection Of Endothelial Cells Via Viral Mirna-Dependent And -Independent Mechanisms, Sara Botto, Jennifer Totonchy, Jean K. Gustin, Ashlee V. Moses

Pharmacy Faculty Articles and Research

Kaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction of heme oxygenase-1 (HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, antiinflammatory, and proliferative activities. Previously published work has shown that KSHV-infected EC in vitro proliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated …

Role Of 5'Tg3'-Interacting Factors (Tgifs) In Vorinostat (Hdac Inhibitor)-Mediated Corneal Fibrosis Inhibition, Ajay Sharma, Nishant R. Sinha, Saad Siddiqui, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

Purpose: We have previously reported that vorinostat, an FDA-approved, clinically used histone deacetylase (HDAC) inhibitor, attenuates corneal fibrosis in vivo in rabbits by blocking transforming growth factor β (TGFβ). The 5′TG3′-interacting factors (TGIFs) are transcriptional repressors of TGFβ1 signaling via the Smad pathway. The present study was designed to explore the expression of TGIFs in human corneal fibroblasts and to investigate their role in mediating the antifibrotic effect of vorinostat.

Methods: Human corneal fibroblast cultures were generated from donor corneas. RNA isolation, cDNA preparation, and PCR were performed to detect the presence of TGIF1 and TGIF2 transcripts. The cultures were …