Digital Commons Network^™

In Vitro Stable Isotope Labeling For Discovery Of Novel Metabolites By Liquid Chromatography-Mass Spectrometry: Confirmation Of Γ-Tocopherol Metabolism In Human A549 Cell, Wen-Chu Yang, Fred E. Regnier, Qing Jiang, Jiri Adamec

Department of Biochemistry: Faculty Publications

A general approach for discovering novel catabolic metabolites from a parent biocompound was developed and validated on metabolism of γ-tocopherol in human A549 cell. Method is based on LC-MS analysis of in vitro stable isotope labeled metabolites and assumes that a parent compound and its metabolites share a common functional group that can be derivatized by well-documented reagents. In this method, two equal aliquots of extracted metabolites are separately derivatized with isotope-coded (heavy) and non-isotope-coded (light) form of derivatizing reagent, mixed at 1:1 ratio and analyzed using LC-MS. The metabolites with common functional group are then easily recognized by determination …

Regulation Of Sealing Ring Formation By L-Plastin And Cortactin In Osteoclasts, Tao Ma, Kavitha Sadashivalah, Nandakumar Madayiputhiya, Meenakshi A. Chellaia

Department of Biochemistry: Faculty Publications

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be …

Structural Basis For Feedback And Pharmacological Inhibition Of Saccharomyces Cerevisiae Glutamate Cysteine Ligase, Ekaterina I Biterova, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A ; R = 19.9%, R_free = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine- …

Cug Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms In Mouse Testes, Maxim Gerashchenko, Dan Su, Vadim Gladyshev

Department of Biochemistry: Faculty Publications

Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate …

Structure Of The Proline Utilization A Proline Dehydrogenase Domain Inactivated By N-Propargylglycine Provides Insight Into Conformational Changes Induced By Substrate Binding And Flavin Reduction, Dhiraj Srivastava, Weidong Zhu, William H. Johnson Jr., Christian P. Whitman, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it …

Analysis Of Leigh Syndrome Mutations In The Yeast Surf1 Homolog Reveals A New Member Of The Cytochrome Oxidase Assembly Factor Family, Megan Bestwick, Mi-Young Jeong, Oleh Khalimonchuk, Hyung Kim, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F²⁴⁹T and Y³⁴⁴D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G¹³⁷E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G¹³⁷E Shy1 mutant phenocopied shy1 Δ cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G137E …

Formation Of The Redox Cofactor Centers During Cox1 Maturation In Yeast Cytochrome Oxidase, Oleh Khalimonchuk, Megan Bestwick, Brigitte Meunier, Talina C. Watts, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The biogenesis of cytochrome c oxidase initiates with synthesis and maturation of the mitochondrionencoded Cox1 subunit prior to the addition of other subunits. Cox1 contains redox cofactors, including the low-spin heme a center and the heterobimetallic heme a₃:Cu_B center. We sought to identify the step in the maturation of Cox1 in which the redox cofactor centers are assembled. Newly synthesized Cox1 is incorporated within one early assembly intermediate containing Mss51 in Saccharomyces cerevisiae. Subsequent Cox1 maturation involves the progression to downstream assembly intermediates involving Coa1 and Shy1. We show that the two heme a cofactor sites …

The Role Of Coa2 In Hemylation Of Yeast Cox1 Revealed By Its Genetic Interaction With Cox10, Megan Bestwick, Oleh Khalimonchuk, Fabien Pierrel, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Saccharomyces cerevisiae cells lacking the cytochrome c oxidase (CcO) assembly factor Coa2 are impaired in Cox1 maturation and exhibit a rapid degradation of newly synthesized Cox1. The respiratory deficiency of coa2 Δ cells is suppressed either by the presence of a mutant allele of the Cox10 farnesyl transferase involved in heme a biosynthesis or through impaired proteolysis by the disruption of the mitochondrial Oma1 protease. Cox10 with an N196K substitution functions as a robust gain-of-function suppressor of the respiratory deficiency of coa2 Δ cells but lacks suppressor activity for two other CcO assembly mutant strains, the coa1 Δ and shy1 …

Quantitative Nuclear Proteomics Identifies Mtor Regulation Of Dna Damage Response, Sricharan Bandhakavi, Young-Mi Kim, Seung-Hyun Ro, Hongwei Xie, Getiria Onsongo, Chang-Bong Jun, Do-Hyung Kim, Timothy J. Griffin

Department of Biochemistry: Faculty Publications

Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed downregulation of nuclear abundance of many proteins involved in translation and RNA modification. …

Methanogenesis By Methanosarcina Acetivorans Involves Two Structurally And Functionally Distinct Classes Of Heterodisulfide Reductase, Nicole R. Buan, William W. Metcalf

Department of Biochemistry: Faculty Publications

Biochemical studies have revealed two distinct classes of Coenzyme B-Coenzyme M heterodisulfide (CoB-S-S-CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methaneproducing archaea. A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane-bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic …

Exon-Primed Intron-Crossing (Epic) Markers For Non-Model Teleost Fishes, Chenhong Li, Jean-Jack M. Riethoven, Lingbo Ma

Papers in Evolution

Background: Exon-primed intron-crossing (EPIC) markers have three advantages over anonymous genomic sequences in studying evolution of natural populations. First, the universal primers designed in exon regions can be applied across a broad taxonomic range. Second, the homology of EPIC-amplified sequences can be easily determined by comparing either their exon or intron portion depending on the genetic distance between the taxa. Third, having both the exon and intron fragments could help in examining genetic variation at the intraspecific and interspecific level simultaneously, particularly helpful when studying species complex. However, the paucity of EPIC markers has hindered multilocus studies using nuclear gene …

Contributions Of Demography And Dispersal Parameters To The Spatial Spread Of A Stage-Structured Insect Invasion, Tom E. X. Miller, Brigitte Tenhumberg

School of Biological Sciences: Faculty Publications

Stage-structured models that integrate demography and dispersal can be used to identify points in the life cycle with large effects on rates of population spatial spread, information that is vital in the development of containment strategies for invasive species. Current challenges in the application of these tools include: (1) accounting for large uncertainty in model parameters, which may violate assumptions of ‘‘local’’ perturbation metrics such as sensitivities and elasticities, and (2) forecasting not only asymptotic rates of spatial spread, as is usually done, but also transient spatial dynamics in the early stages of invasion. We developed an invasion model for …

Effects Of Plant Sex On Range Distributions And Allocation To Reproduction, Marc T.J. Johnson, Stacey Dewitt Smith, Mark D. Rausher

School of Biological Sciences: Faculty Publications

Despite an abundance of theory, few empirical studies have explored the ecological and evolutionary consequences of sex. We used a comparative phylogenetic approach to examine whether transitions between sexual and asexual reproduction are associated with changes in the size and distribution of species’ geographical ranges, and their investment in reproduction.

Here, we reconstructed the phylogeny of the genus Oenothera sections Oenothera and Calylophus (Onagraceae), which contain 35 sexual and 30 functionally asexual species. From each species, we collected data on the geographical distribution and variation in plant traits related to reproduction.

Functionally asexual species occurred at higher latitudes, but did …

Dynamic Changes In Genome-Wide Histone H3 Changes Lysine 4 Methylation Patterns In Response To Dehydration Stress In Arabidopsis Thaliana, Karin V. Van Dijk, Yong Ding, Sridar Achary Malkaram, Jean-Jack Riethoven, Rong Liu, Jingyi Yang, Peter Laczko, Han Chen, Yuannan Xia, Istvan Ladunga, Zoya V. Avramova, Michael E. Fromm

School of Biological Sciences: Faculty Publications

Background: The molecular mechanisms of genome reprogramming during transcriptional responses to stress are associated with specific chromatin modifications. Available data, however, describe histone modifications only at individual plant genes induced by stress. We have no knowledge of chromatin modifications taking place at genes whose transcription has been down-regulated or on the genome-wide chromatin modification patterns that occur during the plant’s response to dehydration stress. Results: Using chromatin immunoprecipitation and deep sequencing (ChIP-Seq) we established the wholegenome distribution patterns of histone H3 lysine 4 mono-, di- and tri-methylation (H3K4me1, H3K4me2, and H3K4me3, respectively) in Arabidopsis thaliana during watered and dehydration stress …

Phosphatidylinositol 5-Phosphate Links Dehydration Stress To The Activity Of Arabidopsis Trithorax-Like Factor Atx1, Ivan Ndamukong, David R. Jones, Hanna Lapko, Nullin Divecha, Zoya V. Avramova

School of Biological Sciences: Faculty Publications

Background: Changes in gene expression enable organisms to respond to environmental stress. Levels of cellular lipid second messengers, such as the phosphoinositide PtdIns5P, change in response to a variety of stresses and can modulate the localization, conformation and activity of a number of intracellular proteins. The plant trithorax factor (ATX1) trimethylates the lysine 4 residue of histone H3 (H3K4me3) at gene coding sequences, which positively correlates with gene transcription. Microarray analysis has identified a target gene (WRKY70) that is regulated by both ATX1 and by the exogenous addition of PtdIns5P in Arabidopsis. Interestingly, ATX1 …

Evolution Of The Kdo2-Lipid A Biosynthesis In Bacteria, Stephen O. Opiyo, Rosevelt L. Pardy, Hideaki Moriyama, Etsuko N. Moriyama

School of Biological Sciences: Faculty Publications

Background: Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo₂-lipid A, the LPS substructure, involves with nine enzymatic steps.

Results: In order to elucidate the evolutionary pathway of Kdo₂-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding …

Variation In Leaf Stomatal Traits Of 28 Tree Species In Relation To Gas Exchange Along An Edaphic Gradient In A Bornean Rain Forest, Sabrina E. Russo, Whitney Logan Cannon, Christian Elowsky, Sylvester Tan, Stuart J. Davies

School of Biological Sciences: Faculty Publications

Premise of the study: Quantifying variation in functional traits associated with shifts in the species composition of plant communities along resource gradients helps identify environmental attributes important for community assembly. Stomates regulate the balance between carbon assimilation and water status in plants. If environmental attributes affecting photosynthetic water-use efficiency govern species distribution along an edaphic gradient, then adaptive variation in stomatal traits of plant species specializing on different soils should reflect belowground resource availability.

Methods: We tested this hypothesis by quantifying stomatal trait variation in understory saplings of 28 Bornean tree species in relation to gas exchange and …

Interspecific Relationships Among Growth, Mortality And Xylem Traits Of Woody Species From New Zealand, Sabrina E. Russo, Kerry L. Jenkins, Susan K. Wiser, Maria Uriarte, Richard P. Duncan, David A. Coomes

School of Biological Sciences: Faculty Publications

1. Wood density is considered a key functional trait influencing the growth and survival of woody plants and has been shown to be related to a slow-fast rate-of-living continuum. Wood density is, however, an emergent trait arising from several vascular properties of wood, including the diameter and frequency of xylem conduits.

2. We aimed to test the hypotheses that there is a set of inter-related trade-offs linked to the different functions of wood, that these trade-offs have direct consequences for tree growth and survival and that these trade-offs underlie the observed correlations between wood density and demographic rates. We evaluated …

Agmatidine, A Modified Cytidine In The Anticodon Of Archaeal TrnaIle, Base Pairs With Adenosine But Not With Guanosine, Debabrata Mandal, Caroline Köhrer, Dan Su, Susan P. Russell, Kady Krivos, Colette M. Castleberry, Paul H. Blum, Patrick A. Limbach, Dieter Söll, Uttam L. Rajbhandary

School of Biological Sciences: Faculty Publications

Modification of the cytidine in the first anticodon position of the AUA decoding tRNA^Ile (tRNA^Ile ₂ ) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui, established its codon reading properties, used liquid chromatography–mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of …

Isolation And Characterization Of Bacteria Resistant To Metallic Copper Surfaces, Christophe Espírito Santo, Paula Vasconcelos Morais, Gregor Grass

School of Biological Sciences: Faculty Publications

Metallic copper alloys have recently attracted attention as a new antimicrobial weapon for areas where surface hygiene is paramount. Currently it is not understood on a molecular level how metallic copper kills microbes, but previous studies have demonstrated that a wide variety of bacteria, including Escherichia coli, Staphylococcus aureus, and Clostridium difficile, are inactivated within minutes or a few hours of exposure. In this study, we show that bacteria isolated from copper alloy coins comprise strains that are especially resistant against the toxic properties exerted by dry metallic copper surfaces. The most resistant of 294 isolates were …

Activity And Toxicity Of Farnesol Towards Candida Albicans Are Dependent On Growth Conditions, Melanie L. Langford, Sahar Hasim, Kenneth W. Nickerson, Audrey L. Atkin

School of Biological Sciences: Faculty Publications

Farnesol interacts with Candida albicans as both a quorum-sensing molecule and toxic agent, but confusion abounds regarding which conditions promote these distinct responses. Farnesol sensitivity was measured when inoculum cell history and size, temperature, and growth media were altered. Parameters for farnesol tolerance/ sensitivity were defined, validating previous studies and identifying new variables, such as energy availability. This study clearly defines what farnesol concentrations are lethal to C. albicans, based on environmental conditions.

Female Field Crickets Incur Increased Parasitism Risk When Near Preferred Song, Cassandra M. Martin, William E. Wagner Jr.

School of Biological Sciences: Faculty Publications

Female animals often prefer males with conspicuous traits because these males provide direct or indirect benefits. Conspicuous male traits, however, can attract predators. This not only increases the risk of predation for conspicuous males but also for the females that prefer them. In the variable field cricket, Gryllus lineaticeps, males that produce preferred song types provide females with greater material benefits, but they are also more likely to attract lethal parasitoid flies. First, we conducted a field experiment that tested the hypothesis that females have a greater risk of fly parasitism when in association with preferred high chirp rate …

Microevolution Of Intermediary Metabolism: Evolutionary Genetics Meets Metabolic Biochemistry, Anthony J. Zera

School of Biological Sciences: Faculty Publications

During the past decade, microevolution of intermediary metabolism has become an important new research focus at the interface between metabolic biochemistry and evolutionary genetics. Increasing recognition of the importance of integrative studies in evolutionary analysis, the rising interest in ‘evolutionary systems biology’, and the development of various ‘omics’ technologies have all contributed significantly to this developing interface. The present review primarily focuses on five prominent areas of recent research on pathway microevolution: lipid metabolism and life-history evolution; the electron transport system, hybrid breakdown and speciation; glycolysis, alcohol metabolism and population adaptation in Drosophila; chemostat selection in microorganisms; and anthocyanin …

Epsvr And Epmeta: Prediction Of Antigenic Epitopes Using Support Vector Regression And Multiple Server Results, Shide Liang, Dandan Zheng, Daron M. Standley, Bo Yao, Martin Zacharias, Chi Zhang

School of Biological Sciences: Faculty Publications

Background: Accurate prediction of antigenic epitopes is important for immunologic research and medical applications, but it is still an open problem in bioinformatics. The case for discontinuous epitopes is even worse - currently there are only a few discontinuous epitope prediction servers available, though discontinuous peptides constitute the majority of all B-cell antigenic epitopes. The small number of structures for antigen-antibody complexes limits the development of reliable discontinuous epitope prediction methods and an unbiased benchmark to evaluate developed methods.

Results: In this work, we present two novel server applications for discontinuous epitope prediction: EPSVR and EPMeta, where EPMeta …

Ignoring Population Structure Can Lead To Erroneous Predictions Of Future Population Size, Brigitte Tenhumberg

School of Biological Sciences: Faculty Publications

Predictions of future population size often assume population structures are stable. Are we missing the boat if we ignore the occurrence of unstable or transient dynamics?

Agricultural crops are regularly infested with insect pests whose population size can reach outbreak densities in some years, causing considerable income loss to farmers. In general, the risk of pest outbreaks is highest when insects disperse to crop fields early in the growing season and the population growth rate is high. The population growth rate measures how much the population size (i.e., number of individuals of a species in a defined area) changes per …

Structured Population Dynamics: An Introduction To Integral Modeling, Joseph Briggs, Kathryn Dabbs, Michael Holm, Joan Lubben, Richard Rebarber, Brigitte Tenhumberg, Daniel Riser-Espinoza

School of Biological Sciences: Faculty Publications

In this article we illuminate the differences and similarities between matrix population models and integral population models for single-species stage structured populations. We illustrate the use of integral models with an application to Platte thistle, following Rose et al. [22], showing how the model is determined by basic life history functions. PPMs are ubiquitous in ecology, but for many purposes an IPM might be easier and/or more accurate to use. In TABLE 1 we summarize the similarities between PPMs and IPMs. In order to compare the predictions for PPMs and IPMs, enough data must be available to find the parameters …

Analysis Of Mirna Modifications, Bin Yu, Xuemei Chen

School of Biological Sciences: Faculty Publications

After transcription, a large number of cellular RNAs employ modifications to increase their diversity and functional potential. Modifications can occur on the base, ribose, or both, and are important steps in the maturation of many RNAs. Our lab recently showed that plant microRNAs (miRNAs) possess a 2′-O-methyl group on the ribose of the 3′ terminal nucleotide, and that this methyl group is added after miRNA/miRNA* formation. One function of this modification is to protect miRNAs from 3′ terminal uridylation by an unknown enzymatic activity. It is possible that uridylation of miRNAs triggers their degradation. Here we describe a …

Bioassays Of Compounds With Potential Juvenoid Activity On Drosophila Melanogaster: Juvenile Hormone Iii, Bisepoxide Juvenile Hormone Iii, And Methyl Farnesoates, Lawrence G. Harshman, Ki-Duck Song, Josephina Casas, A. Schuurmans, Eichii Kuwano, Stephen D. Kachman, Lynn M. Riddiford, Bruce D. Hammock

Lawrence G. Harshman Publications

Metabolites of the 6,7,10,11 bisepoxide juvenile hormone III (JHB₃), and other potential juvenoids, were tested for juvenile hormone activity using early instar or early stage pupae of Drosophila mela-nogaster. Importantly, methyl farnesoates were tested as they might have JH-like activity on Dipteran juveniles. Larvae were exposed to compounds in medium, or the compounds were applied to white puparia. In the assays employed in the present study, there was no indication for JH activity associ-ated with the metabolites of JHB₃. The activity of methyl farnesoate (MF) was higher than that of JH III and far greater …

A Signal-Substrate Match In The Substrate-Borne Component Of A Multimodal Courtship Display, Damian O. Elias, Andrew C. Mason, Eileen Hebets

Eileen Hebets Publications

The environment can impose strong limitations on the efficacy of signal transmission. In particular, for vibratory communication, the signaling environment is often extremely heterogeneous at small scales. Nevertheless, natural selection is expected to select for signals well-suited for effective transmission. Here, we test for substrate-dependent signal efficacy in the wolf spider Schizocosa stridulans Stratton 1991. We first explore the transmission characteristics of this important signaling modality by playing recorded substrate-borne signals through three different substrates (leaf litter, pine litter, and red clay) and measuring the propagated signal. We found that the substrate-borne signal of S. stridulans attenuates the least on …

Multimodal Courtship Efficacy Of Schizocosa Retrorsa Wolf Spiders: Implications Of An Additional Signal Modality, Aaron S. Rundus, Roger D. Santer, Eileen Hebets

Eileen Hebets Publications

Here, we simultaneously examine both content and efficacy-based sources of selection on the visual and seismic multimodal courtship display of the wolf spider Schizocosa retrorsa. Immature field-collected S. retrorsa were reared in the laboratory on either high-quantity diet (HD) or low-quantity diet (LD) treatments. On maturation, females of each diet treatment were run in simultaneous mate choice trials with both a HD and an LD male (content-based selection). Simultaneous mate choice trials were conducted across different signaling environments (efficacy-based selection) in a fully crossed 2 × 2 design with visual treatments of light/dark (visual signal present/absent) and seismic treatments …