Biotechnology Commons^™

Oxidative Stress, Metabolomics Profiling, And Mechanism Of Local Anesthetic Induced Cell Death In Yeast, Cory Honsinger Thomas Boone, Ryan A. Grove, Dana Adamcova, Javier Seravalli, Jiri Adamec

Department of Biochemistry: Faculty Publications

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6 h. Exposure of an LC₅₀ dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by …

Toxoplasma Dj-1 Regulates Organelle Secretion By A Direct Interaction With Calcium-Dependent Protein Kinase 1, Matthew A. Child, Megan Garland, Ian Foe, Peter Madzelan, Moritz Treeck, Wouter A. Van Der Linden, Kristina Oresic Bender, Evanthie Weerapana, Mark A. Wilson, John C. Boothroyd, Michael L. Reese

Department of Biochemistry: Faculty Publications

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-A resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, …

Seasonal Switchgrass Ecotype Contributions To Soil Organic Carbon, Deep Soil Microbial Community Composition And Rhizodeposit Uptake During An Extreme Drought, Catherine E. Stewart, Damaris Roosendaal, Karolien Denef, Elizabeth Pruessner, Louise H. Comas, Gautam Sarath, Virginia L. Jin, Marty R. Schmer, Madhavan Soundararajan

Department of Biochemistry: Faculty Publications

The importance of rhizodeposit C and associated microbial communities in deep soil C stabilization is relatively unknown. Phenotypic variability in plant root biomass could impact C cycling through belowground plant allocation, rooting architecture, and microbial community abundance and composition. We used a pulse-chase ¹³C labeling experiment with compound-specific stable-isotope probing to investigate the importance of rhizodeposit C to deep soil microbial biomass under two switchgrass ecotypes (Panicum virgatum L., Kanlow and Summer) with contrasting root morphology. We quantified root phenology, soil microbial biomass (phospholipid fatty acids, PLFA), and microbial rhizodeposit uptake (¹³C-PLFAs) to 150 cm over …

Secs (Sinusoidal Endothelial Cells), Liver Microenvironment, And Fibrosis, Vaishaali Natarajan, Edward N. Harris, Srivatsan Kidambi

Department of Biochemistry: Faculty Publications

Liver fibrosis is awound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs …

Gene Duplication And Neo-Functionalization In The Evolutionary And Functional Divergence Of The Metazoan Copper Transporters Ctr1 And Ctr2, Brandon L. Logeman, L. Kent Wood, Jaekwoon Lee, Dennis J. Thiele

Department of Biochemistry: Faculty Publications

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu_ across both the plasma membrane and endomembranes. Although mammalian Ctr¹ functions as a Cu_ transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr², has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr¹ activity, but …

Carbon And Acyl Chain Flux During Stress-Induced Triglyceride Accumulation By Stable Isotopic Labeling Of The Polar Microalga Coccomyxa Subellipsoidea C169, James W. Allen, Concetta C. Dirusso, Paul N. Black

Department of Biochemistry: Faculty Publications

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO₂ balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-¹³C]glucose, ¹³CO₂, or D₂O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid …

Quantification Of Cell Signaling Networks Using Kinase Activity Chemosensors, Jon R. Beck, Edward N. Harris, Cliff I. Stains

Department of Biochemistry: Faculty Publications

The ability to directly determine endogenous kinase activity in tissue homogenates provides valuable insights into signaling aberrations that underlie disease phenotypes. When activity data is collected across a panel of kinases, a unique “signaling fingerprint” is generated that allows for discrimination between diseased and normal tissue. Here we describe the use of peptide-based kinase activity sensors to fingerprint the signaling changes associated with disease states. This approach leverages the phosphorylation-sensitive sulfonamido-oxine (Sox) fluorophore to provide a direct readout of kinase enzymatic activity in unfractionated tissue homogenates from animal models or clinical samples. To demonstrate the application of this technology, we …

Phylogenomic Analysis Of Copepoda (Arthropoda, Crustacea) Reveals Unexpected Similarities With Earlier Proposed Morphological Phylogenies, Seong-Il Eyun

Nebraska Center for Biotechnology: Faculty and Staff Publications

Background: Copepods play a critical role in marine ecosystems but have been poorly investigated in phylogenetic studies. Morphological evidence supports the monophyly of copepods, whereas interordinal relationships continue to be debated. In particular, the phylogenetic position of the order Harpacticoida is still ambiguous and inconsistent among studies. Until now, a small number of molecular studies have been done using only a limited number or even partial genes and thus there is so far no consensus at the order-level.

Results: This study attempted to resolve phylogenetic relationships among and within four major copepod orders including Harpacticoida and the phylogenetic position of …

Evolutionary History Of Chemosensory-Related Gene Families Across The Arthropoda, Seong-Il Eyun, Ho Young Soh, Marijan Posavi, James B. Munro, Daniel S.T. Hughes, Shwetha C. Murali, Jiaxin Qu, Shannon Dugan, Sandra L. Lee, Hsu Chao, Huyen Dinh, Yi Han, Harshavardhan Doddapaneni, Kim C. Worley, Donna M. Muzny, Eun-Ok Park, Joana C. Silva, Richard A. Gibbs, Stephen Richards, Carol Eunmi Lee

Nebraska Center for Biotechnology: Faculty and Staff Publications

Chemosensory-related gene (CRG) families have been studied extensively in insects, but their evolutionary history across the Arthropoda had remained relatively unexplored. Here, we address current hypotheses and prior conclusions on CRG family evolution using a more comprehensive data set. In particular, odorant receptors were hypothesized to have proliferated during terrestrial colonization by insects (hexapods), but their association with other pancrustacean clades and with independent terrestrial colonizations in other arthropod subphyla have been unclear. We also examine hypotheses on which arthropod CRG family is most ancient. Thus, we reconstructed phylogenies of CRGs, including those from new arthropod genomes and transcriptomes, and …

A Method For Measuring The Attachment Strength Of The Cestode Hymenolepis Diminuta To The Rat Intestine, Wanchuan Xie, Gábor R. Rácz, Benjamin S. Terry, Scott Lyell Gardner

Scott L. Gardner Publications

A unique adaptation of many internal parasites of mammals is their ability to stay in the intestine for extended periods of time and resist the normal peristaltic movements and forces that push and expel material. To better understand parasite adhesion behavior and replicate their attachment method in medical devices, an experiment was designed and performed using the rat tapeworm, Hymenolepis diminuta. The experiment employed a tensile test machine and a digital scale and was designed to calculate the attachment strength of the scolex to the mucosa through the change of the value of the digital scale during the tensile …

The Intestinal Copper Exporter Cua-1 Is Required For Systemic Copper Homeostasis In Caenorhabditis Elegans, Haarin Chun, Anuj Kumar Sharma, Jaekwon Lee, Jefferson Chan, Shang Jia, Byung-Eun Kim

Department of Biochemistry: Faculty Publications

Copper plays key catalytic and regulatory roles in biochemical processes essential for normal growth, development, and health. Defects in copper metabolism cause Menkes and Wilson’s disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of copper. The intestinal enterocyte is a key regulatory point for copper absorption into the body; however, the mechanisms by which intestinal cells transport copper to maintain organismal copper homeostasis are poorly understood. Here, we identify a mechanism by which organismal copper homeostasis is maintained by …

Assessing The Risk Of Resistance Evolution, Adult And Larval Susceptibility, And Sublethal Effects After Exposure Of Corn Rootworms To Vacuolar Atpase-A And Snf7 Dsrnas, Adriano Elias Pereira

Department of Entomology: Dissertations, Theses, and Student Research

Corn rootworms are Diabroticite beetles from the genus Diabrotica and include the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, and the northern corn rootworm, D. barberi Smith & Lawrence, as the most important pests in the U.S. Corn Belt. The southern corn rootworm (SCR), D. undecimpunctata howardi Barber, is considered a polyphagous plant pest and feeds in a wide variety of plants including peanuts, cucurbits, soybeans, and especially corn. Resistance to control methods including behavioral resistance to crop rotation and to corn hybrids expressing Bacillus thuringiensis Berliner toxins in WCR has frequently been reported. SCR has been considered a …

Structural Determinants Allowing Transferase Activity In Sensitive To Freezing 2, Classified As A Family I Glycosyl Hydrolase, Rebecca Roston, Kun Wang, Leslie A. Kuhn, Christoph Benning

Department of Biochemistry: Faculty Publications

Background: SENSITIVE TO FREEZING 2 (SFR2) is classified as a glycosyl hydrolase, and by using glycosyltransferase activity, it modifies membrane lipids to promote freeze tolerance.

Results: Although the active site of SFR2 is identical to hydrolases, adjacent loop regions contribute to its transferase activity.

Conclusion: Transferase activity evolved by modifications external to the core catalytic site.

Significance: Defined structure-function relationships will inform engineering of transferases and freeze tolerance.

Sending Out An Sos: Mitochondria As A Signaling Hub, Iryna Bohovych, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Normal cellular physiology is critically dependent on numerous mitochondrial activities including energy conversion, cofactor and precursor metabolite synthesis, and regulation of ion and redox homeostasis. Advances in mitochondrial research during the last two decades provide solid evidence that these organelles are deeply integrated with the rest of the cell and multiple mechanisms are in place to monitor and communicate functional states of mitochondria. In many cases, however, the exact molecular nature of various mitochondria-to-cell communication pathways is only beginning to emerge. Here, we review various signals emitted by distressed or dysfunctional mitochondria and the stress-responsive pathways activated in response to …

Loss Of Exogenous Androgen Dependence By Prostate Tumor Cells Is Associated With Elevated Glucuronidation Potential, Brenna M. Zimmer, Michelle E. Howell, Qin Wei, Linlin Ma, Trevor Romsdahl, Eileen G. Loughman, Jennifer E. Markham, Javier Seravalli, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD⁺-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate, and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen dependent versus castration resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration resistant tumor cells expressed higher …

Small-Angle X-Ray Scattering Studies Of The Oligomeric State And Quaternary Structure Of The Trifunctional Proline Utilization A (Puta) Flavoprotein From Escherichia Coli, Ranjan K. Singh, John D. Larson, Weidong Zhu, Robert P. Rambo, Greg L. Hura, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Background: Trifunctional proline utilization A (PutA) proteins are multifunctional flavoproteins that catalyze two reactions and repress transcription of the put regulon.

Results: PutA from Escherichia coli is a V-shaped dimer, with the DNA-binding domain mediating dimerization.

Conclusion: Oligomeric state and quaternary structures are not conserved by PutAs.

Significance: The first three-dimensional structural information for any trifunctional PutA is reported.

Determining Gene Specific Chromatin Differences In Sulfolobus Solfataricus: Expression Of Merr Protein For Targeted-Chip Antibody Production, Erica M. North, Sophie Payne, Sam Mccarthy, Tyler Johnson, Paul H. Blum

UCARE: Research Products

In this project the repressor protein MerR from the Sulfolobus solfataricus mercury resistance operon was cloned into pET28b and transformed into Roetta 2 E.coli strains for overexpression and purification. Large quantities of recombinant MerR will be used for subsequent injection into a mammalian host for antibody production. These antibodies will be used in targeted-ChIP studies in which gene specific chromatin modification states will be analyzed. The overproduction of MerR is part of a larger project where future research could produce data on whether gene expression levels and chromatin modifiation states could be correlated at an individual gene level, possibly suggesting …