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Altering Oligomerization Of Epha2 Via Mutations In The Intracellular Domain, Ryan W. Lingerak 2018 The University of Akron

Altering Oligomerization Of Epha2 Via Mutations In The Intracellular Domain, Ryan W. Lingerak

Honors Research Projects

Eph receptor tyrosine kinases (RTKs) are activated by membrane-bound ligands called ephrins. Eph RTKs are divided into two subclasses, each activated by a specific classes of the ligand ephrin. The overexpression of Eph receptors is correlated to cancer cell metastasis in several different types of cancers. Studies with the EphA2 extracellular domain (ECD) and ephrinA1 ligand have shown that upon binding of ephrin to the receptor, EphA2 undergoes increased oligomerization and activation. This indicates that oligomerization is intimately connected to kinase activity. High resolution crystal structures of the EphA2 ECD have revealed some details of these ligand bound oligomers, as ...


Tea Induction Of Cyp6a8 Promoter In Drosophila Melanogaster, Fiona Retzer 2017 University of Tennessee, Knoxville

Tea Induction Of Cyp6a8 Promoter In Drosophila Melanogaster, Fiona Retzer

University of Tennessee Honors Thesis Projects

No abstract provided.


Attenuation Of Na/K-Atpase Mediated Oxidant Amplification With Pnaktide Ameliorates Experimental Uremic Cardiomyopathy, Jiang Liu, Jiang Tian, Muhammad Chaudhry, Nader G. Abraham, Joseph I Shapiro 2017 New York Medical College

Attenuation Of Na/K-Atpase Mediated Oxidant Amplification With Pnaktide Ameliorates Experimental Uremic Cardiomyopathy, Jiang Liu, Jiang Tian, Muhammad Chaudhry, Nader G. Abraham, Joseph I Shapiro

Nader G. Abraham

We have previously reported that the sodium potassium adenosine triphosphatase (Na/K-ATPase) can effect the amplification of reactive oxygen species. In this study, we examined whether attenuation of oxidant stress by antagonism of Na/K-ATPase oxidant amplification might ameliorate experimental uremic cardiomyopathy induced by partial nephrectomy (PNx). PNx induced the development of cardiac morphological and biochemical changes consistent with human uremic cardiomyopathy. Both inhibition of Na/K-ATPase oxidant amplification with pNaKtide and induction of heme oxygenase-1 (HO-1) with cobalt protoporphyrin (CoPP) markedly attenuated the development of phenotypical features of uremic cardiomyopathy. In a reversal study, administration of pNaKtide after the ...


Molecular Mechanisms Of Mycoplasma Hyopneumoniae Adherence To Swine Respiratory Epithelial Cells, Qijing Zhang 2017 Iowa State University

Molecular Mechanisms Of Mycoplasma Hyopneumoniae Adherence To Swine Respiratory Epithelial Cells, Qijing Zhang

Qijing Zhang

A microtiter plate adherence assay for Mycoplasma hyopneumoniae was established by using purified swine tracheal cilia which are the natural targets for the mycoplasma. M. hyopneumoniae bound specifically to solubilized cilia immobilized onto microtiter plates. Dextran sulfate, heparin, chondroitin sulfate, laminin, mucin, and fucoidan significantly inhibited binding of the mycoplasmas to cilia. Heparin, mucin, fucoidan, and chondroitin sulfate interacted with the adhesins on the surface of mycoplasmas, whereas laminin blocked the receptors in cilia. Treatment of cilia with neuraminidase appeared to promote adherence of the mycoplasmas; whereas, treatment of cilia with sodium metaperiodate decreased the binding. In the second study ...


Differential Surface Stress Sensor For Detection Of Chemical And Biological Species, K. Kang, Marit Nilsen-Hamilton, Pranav Shrotriya 2017 Iowa State University

Differential Surface Stress Sensor For Detection Of Chemical And Biological Species, K. Kang, Marit Nilsen-Hamilton, Pranav Shrotriya

Pranav Shrotriya

We report a sensor consisting of two micromachined cantilevers (a sensing/reference pair) that is suitable for detection of chemical and biological species. The sensing strategy involves coating the sensing cantilever with receptors that have high affinities for the analyte. The presence of analyte is detected by determining the differential surface stress associated with its adsorption/absorption to the sensing cantilever. An interferometric technique is utilized to measure the differential bending of the sensing cantilever with respect to reference. Surface stress associated with hybridization of single stranded DNA is measured to demonstrate the unique advantages of the sensor.


Aptamer Functionalized Microcantilever Sensors For Cocaine Detection, Kyungho Kang, Ashish Sachan, Marit Nilsen-Hamilton, Pranav Shrotriya 2017 Iowa State University

Aptamer Functionalized Microcantilever Sensors For Cocaine Detection, Kyungho Kang, Ashish Sachan, Marit Nilsen-Hamilton, Pranav Shrotriya

Pranav Shrotriya

A cocaine-specific aptamer was used as a receptor molecule in a microcantilever-based surface stress sensor for detection of cocaine molecules. An interferometric technique that relies on measuring differential displacement between two microcantilevers (a sensing/reference pair) was utilized to measure the cocaine/aptamer binding induced surface stress changes. Sensing experiments were performed for different concentrations of cocaine from 25 to 500 μM in order to determine the sensor response as a function of cocaine concentration. In the lower concentration range from 25 to 100 μM, surface stress values increased proportionally to coverage of aptamer/cocaine complexes from 11 to 26 ...


An Rna Aptamer-Based Microcantilever Sensor To Detect The Inflammatory Marker, Mouse Lipocalin-2, Lijie Zhai, Tianjiao Wang, Kyungho Kang, Yue Zhao, Pranav Shrotriya, Marit Nilsen-Hamilton 2017 Iowa State University

An Rna Aptamer-Based Microcantilever Sensor To Detect The Inflammatory Marker, Mouse Lipocalin-2, Lijie Zhai, Tianjiao Wang, Kyungho Kang, Yue Zhao, Pranav Shrotriya, Marit Nilsen-Hamilton

Pranav Shrotriya

Lipocalin-2 (Lcn2) is a biomarker for many inflammatory-based diseases, including acute kidney injury, cardiovascular stress, diabetes, and various cancers. Inflammatory transitions occur rapidly in kidney and cardiovascular disease, for which an in-line monitor could be beneficial. Microcantilever devices with aptamers as recognition elements can be effective and rapidly responsive sensors. Here, we have selected and characterized an RNA aptamer that specifically binds mouse Lcn2 (mLcn2) with a dissociation constant of 340 ± 70 nM in solution and 38 ± 22 nM when immobilized on a surface. The higher apparent affinity of the immobilized aptamer may result from its effective multivalency that decreases ...


Safety And Potential Efficacy Of Gemfibrozil As A Supportive Treatment For Children With Late Infantile Neuronal Ceroid Lipofuscinosis And Other Lipid Storage Disorders., Kyeongsoon Kim, Hynda K Kleinman, Hahn-Jun Lee, Kalipada Pahan 2017 George Washington University

Safety And Potential Efficacy Of Gemfibrozil As A Supportive Treatment For Children With Late Infantile Neuronal Ceroid Lipofuscinosis And Other Lipid Storage Disorders., Kyeongsoon Kim, Hynda K Kleinman, Hahn-Jun Lee, Kalipada Pahan

Biochemistry and Molecular Medicine Faculty Publications

Neuronal Ceroid Lipofuscinosis (NCL), also known as Batten disease, is a group of genetically distinct lysosomal disorders that mainly affect the central nervous system, resulting in progressive motor and cognitive decline primarily in children. Multiple distinct genes involved in the metabolism of lipids have been identified to date with various mutations in this family of diseases. There is no cure for these diseases but some new therapeutic approaches have been tested that offer more hope than the standard palliative care. Many of the therapeutic advances require invasive procedures but some progress in slowing the disease has been found and more ...


Parsing A Multifunctional Biosynthetic Gene Cluster From Rice: Biochemical Characterization Of Cyp71z6 & 7, Yinsheng Wu, Matthew L. Hillwig, Qiang Wang, Reuben J. Peters 2017 Iowa State University

Parsing A Multifunctional Biosynthetic Gene Cluster From Rice: Biochemical Characterization Of Cyp71z6 & 7, Yinsheng Wu, Matthew L. Hillwig, Qiang Wang, Reuben J. Peters

Reuben J. Peters

Rice (Oryza sativa) contains a biosynthetic gene cluster associated with production of at least two groups of diterpenoid phytoalexins, the antifungal phytocassanes and antibacterial oryzalides. While cytochromes P450 (CYP) from this cluster are known to be involved in phytocassane production, such mono-oxygenase activity relevant to oryzalide biosynthesis was unknown. Here we report biochemical characterization demonstrating that CYP71Z6 from this cluster acts as an ent-isokaurene C2-hydroxylase that is presumably involved in the biosynthesis of oryzalides. Our results further suggest that the closely related and co-clustered CYP71Z7 likely acts as a C2- hydroxylase involved in a latter step of phytocassane biosynthesis. Thus ...


Rv0989c Encodes A Novel (E)-Geranyl Diphosphate Synthase Facilitating Decaprenyl Diphosphate Biosynthesis In Mycobacterium Tuberculosis, Francis M. Mann, Jill A. Thomas, Reuben J. Peters 2017 Iowa State University

Rv0989c Encodes A Novel (E)-Geranyl Diphosphate Synthase Facilitating Decaprenyl Diphosphate Biosynthesis In Mycobacterium Tuberculosis, Francis M. Mann, Jill A. Thomas, Reuben J. Peters

Reuben J. Peters

Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the “missing” GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from ...


Insights Into Diterpene Cyclization From Structure Of Bifunctional Abietadiene Synthase From Abies Grandis, Ke Zhou, Yang Gao, Julie A. Hoy, Francis M. Mann, Richard B. Honzatko, Reuben J. Peters 2017 Iowa State University

Insights Into Diterpene Cyclization From Structure Of Bifunctional Abietadiene Synthase From Abies Grandis, Ke Zhou, Yang Gao, Julie A. Hoy, Francis M. Mann, Richard B. Honzatko, Reuben J. Peters

Reuben J. Peters

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of ...


Isotuberculosinol: The Unusual Case Of An Immunomodulatory Diterpenoid From Mycobacterium Tuberculosis, Francis M. Mann, Reuben J. Peters 2017 Winona State University

Isotuberculosinol: The Unusual Case Of An Immunomodulatory Diterpenoid From Mycobacterium Tuberculosis, Francis M. Mann, Reuben J. Peters

Reuben J. Peters

The actinomycetes family of bacteria is classically considered a rich source of natural products [1]. It is often suggested that such compounds have been selected by evolution to have biological activity [2]. For example, given the microbial-rich soil environment of the actinomycetes native habitat, their natural products have been suggested to serve in chemical warfare and/or signaling, which has been further connected to their leading role in providing pharmaceutically relevant antibiotic agents [3]. Nevertheless, surprisingly few of these molecules have been definitively assigned specific physiological roles in the native producing bacteria.


Two Rings In Them All: The Labdane-Related Diterpenoids, Reuben J. Peters 2017 Iowa State University

Two Rings In Them All: The Labdane-Related Diterpenoids, Reuben J. Peters

Reuben J. Peters

In his original exposition of the biogenetic isoprenoid rule, L. Ruzicka noted the structural identity between the fused A/B rings of triterpenoids/sterols and certain multicyclic diterpenoids as part of the impetus leading to that profound insight. His prescient hypothesis that this chemical structure relationship reflects similarities in the initial cyclization of these diterpenoids with that occurring in triterpenoid biosynthesis has since been verified. However, this chemical structure relationship does not continue to hold true for the additional rings found in many of these di- and tri- terpenoid natural products. This is now appreciated to arise from differences in ...


Magnesium Depletion Triggers Production Of An Immune Modulating Diterpenoid In Mycobacterium Tuberculosis, Francis M. Mann, Brian C. VanderVen, Reuben J. Peters 2017 Iowa State University

Magnesium Depletion Triggers Production Of An Immune Modulating Diterpenoid In Mycobacterium Tuberculosis, Francis M. Mann, Brian C. Vanderven, Reuben J. Peters

Reuben J. Peters

Mycobacterium tuberculosis (Mtb) is the causative agent of the human disease Tuberculosis, and remains a worldwide health threat responsible for ~1.7 million deaths annually. During infection, Mtb prevents acidification of the engulfing phagosome, thus blocking endocytic progression and eventually leading to stable residence. The diterpenoid metabolite isotuberculosinol (isoTb) exhibits biological activity indicative of a role in this early arrest of phagosome maturation. Presumably, isoTb production should be induced by phagosomal entry. However, the relevant enzymatic genes are not transcriptionally upregulated during engulfment. Previous examination of the initial biosynthetic enzyme (Rv3377c/MtHPS) involved in isoTb biosynthesis revealed striking inhibition by ...


Structure And Mechanism Of The Diterpene Cyclase Ent-Copalyl Diphosphate Synthase, Mustafa Köksal, Huayou Hu, Robert M. Coates, Reuben J. Peters, David D. Christianson 2017 University of Pennsylvania

Structure And Mechanism Of The Diterpene Cyclase Ent-Copalyl Diphosphate Synthase, Mustafa Köksal, Huayou Hu, Robert M. Coates, Reuben J. Peters, David D. Christianson

Reuben J. Peters

The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.


Terpenoid Synthase Structures: A So Far Incomplete View Of Complex Catalysis, Yang Gao, Richard B. Honzatko, Reuben J. Peters 2017 Iowa State University

Terpenoid Synthase Structures: A So Far Incomplete View Of Complex Catalysis, Yang Gao, Richard B. Honzatko, Reuben J. Peters

Reuben J. Peters

The complexity of terpenoid natural products has drawn significant interest, particularly since their common (poly)isoprenyl origins were discovered. Notably, much of this complexity is derived from the highly variable cyclized and/or rearranged nature of the observed hydrocarbon skeletal structures. Indeed, at least in some cases it is difficult to immediately recognize their derivation from poly-isoprenyl precursors. Nevertheless, these diverse structures are formed by sequential elongation to acyclic precursors, most often with subsequent cyclization and/or rearrangement. Strikingly, the reactions used to assemble and diversify terpenoid backbones share a common carbocationic driven mechanism, although the means by which the ...


Cyp99a3: Functional Identification Of A Diterpene Oxidase From The Momilactone Biosynthetic Gene Cluster In Rice, Qiang Wang, Matthew L. Hillwig, Reuben J. Peters 2017 Iowa State University

Cyp99a3: Functional Identification Of A Diterpene Oxidase From The Momilactone Biosynthetic Gene Cluster In Rice, Qiang Wang, Matthew L. Hillwig, Reuben J. Peters

Reuben J. Peters

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochromes P450 mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNAi double knock-down of this pair of closely related CYP reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which ultimately was achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that, while CYP99A2 does not exhibit significant ...


Characterization Of Cyp76m5–8 Indicates Metabolic Plasticity Within A Plant Biosynthetic Gene Cluster, Qiang Wang, Matthew L. Hillwig, Kazunori Okada, Kohei Yamazaki, Yisheng Wu, Sivakumar Swaminathan, Hisakazu Yamane, Reuben J. Peters 2017 Iowa State University

Characterization Of Cyp76m5–8 Indicates Metabolic Plasticity Within A Plant Biosynthetic Gene Cluster, Qiang Wang, Matthew L. Hillwig, Kazunori Okada, Kohei Yamazaki, Yisheng Wu, Sivakumar Swaminathan, Hisakazu Yamane, Reuben J. Peters

Reuben J. Peters

Recent reports have revealed genomic clustering of enzymatic genes for particular biosynthetic pathways in plant specialized/secondary metabolism. Rice (Oryza sativa) carries two such clusters for production of antimicrobial diterpenoid phytoalexins, with the cluster on chromosome 2 containing four closely related/homologous members of the cytochrome P450 CYP76M subfamily (CYP76M5–8). Notably, the underlying evolutionary expansion of these CYP appears to have occurred after assembly of the ancestral biosynthetic gene cluster, suggesting separate roles. It has been demonstrated that CYP76M7 catalyzes C11α-hydroxylation of ent-cassadiene, and presumably mediates an early step in biosynthesis of the derived phytocassane class of phytoalexins. Here ...


Functional Characterization Of Wheat Copalyl Diphosphate Synthases Sheds Light On The Early Evolution Of Labdane-Related Diterpenoid Metabolism In The Cereals, Yisheng Wu, Ke Zhou, Tomonobu Toyomasu, Chizu Sugawara, Madoka Oku, Shiho Abe, Masami Usui, Wataru Mitsuhashi, Makiko Chono, Peter M. Chandler, Reuben J. Peters 2017 Iowa State University

Functional Characterization Of Wheat Copalyl Diphosphate Synthases Sheds Light On The Early Evolution Of Labdane-Related Diterpenoid Metabolism In The Cereals, Yisheng Wu, Ke Zhou, Tomonobu Toyomasu, Chizu Sugawara, Madoka Oku, Shiho Abe, Masami Usui, Wataru Mitsuhashi, Makiko Chono, Peter M. Chandler, Reuben J. Peters

Reuben J. Peters

Two of the most agriculturally important cereal crop plants are wheat (Triticum aestivum) and rice (Oryza sativa). Rice has been shown to produce a number of diterpenoid natural products as phytoalexins and/or allelochemicals – specifically, labdane-related diterpenoids, whose biosynthesis proceeds via formation of an eponymous labdadienyl/copalyl diphosphate (CPP) intermediate (e.g., the ent-CPP of gibberellin phytohormone biosynthesis). Similar to rice, wheat encodes a number of CPP synthases (CPS), and the three CPS characterized to date (TaCPS1,2,&3) all have been suggested to produce ent-CPP. However, several of the downstream diterpene synthases will only react with CPP intermediate of normal or syn, but not ent, stereochemistry, as described in the accompanying report. Investigation of additional CPS did not resolve this issue, as the only other functional synthase (TaCPS4) also produced ent-CPP. Chiral product characterization of all the TaCPS then revealed that TaCPS2 uniquely produces normal, rather than ent-, CPP; thus, providing a suitable substrate source for the downstream diterpene synthases. Notably, TaCPS2 is most homologous to the similarly stereochemically differentiated syn-CPP ...


A Single Residue Switch For Mg2+-Dependent Inhibition Characterizes Plant Class Ii Diterpene Cyclases From Primary And Secondary Metabolism, Francis M. Mann, Sladjana Prisic, Emily K. Davenport, Mara K. Determan, Robert M. Coates, Reuben J. Peters 2017 Iowa State University

A Single Residue Switch For Mg2+-Dependent Inhibition Characterizes Plant Class Ii Diterpene Cyclases From Primary And Secondary Metabolism, Francis M. Mann, Sladjana Prisic, Emily K. Davenport, Mara K. Determan, Robert M. Coates, Reuben J. Peters

Reuben J. Peters

Class II diterpene cyclases mediate the acid-initiated cycloisomerization reaction that serves as the committed step in biosynthesis of the large class of labdane-related diterpenoid natural products, which includes the important gibberellin plant hormones. Intriguingly, these enzymes are differentially susceptible to inhibition by their Mg2+ cofactor, with those involved in gibberellin biosynthesis being more sensitive to such inhibition than those devoted to secondary metabolism, which presumably limits flux toward the potent gibberellin phytohormones. Such inhibition has been suggested to arise from intrasteric Mg2+ binding to the DXDD motif that cooperatively acts as the catalytic acid, whose affinity must then be modulated ...


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