Biotechnology Commons^™

Isolation And Characterization Of Galactinol Synthases From Hybrid Poplar, Faride Unda, Thomas Canam, Lindsay Preston, Shawn Mansfield

Faculty Research & Creative Activity

The raffinose family of oligosaccharides (RFOs) serve as transport carbohydrates in the phloem, storage compounds in sink tissues, and putative biological agents to combat both abiotic and biotic stress in several plant species. To investigate further the functional roles of this class of compounds in trees, two cDNAs encoding galactinol synthase (GolS, EC 2.4.1.123), which catalyses the first step in the biosynthesis of RFOs, were identified and cloned from hybrid poplar (Populus alba3grandidentata). Phylogenetic analyses of the Populus GolS isoforms with other known GolS proteins suggested a putative role for these enzymes during biotic or abiotic stress in hybrid poplar. …

Nutraceutical Antioxidants And Their Therapeutic Potential In Neurodegeneration, Erika Kristine Ross

Electronic Theses and Dissertations

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease that affects motor neurons of the brain and spinal cord. Many studies indicate that mitochondrial oxidative stress (MOS) is a principal mechanism underlying the pathophysiology of this and other devastating neurodegenerative diseases. Here, we investigated a unique whey protein supplement (Immunocal®) to determine its neuroprotective efficacy in several in vitro models of MOS and in an in vivo mouse model of ALS. This non-denatured whey supplement contains cystine which is an oxidized form of cysteine, an essential precursor for synthesis of the endogenous antioxidant, glutathione (GSH). In primary cultured rat cerebellar …

A Novel Link Between Akt1 And Twist1 In Ovarian Tumor Cell Motility And Invasiveness, Nirav Shah

Electronic Theses and Dissertations

Ovarian cancer results in more deaths per year than any other cancer of the female reproductive system. The low survival rate is partly due to the lack of early detection and the susceptibility to relapse. The AKT serine threonine kinase plays a pivotal role in hallmark cellular processes for the progression of ovarian cancer, including tumor cell growth and migration. Therapeutic targeting of pan-AKT has been problematic, in part due to feedback mechanisms and crosstalk with other pathways. The hypothesis for this study is that AKT 1, -2 and -3 isoforms may have different roles and regulate cell processes in …

The Cell Collective: Toward An Open And Collaborative Approach To Systems Biology, Tomáš Helikar, Bryan Kowal, Sean Mcclenathan, Mitchell Bruckner, Thaine Rowley, Alex Madrahimov, Ben Wicks, Manish Shrestha, Kahani Limbu, Jim A. Rogers

Department of Biochemistry: Faculty Publications

Background: Despite decades of new discoveries in biomedical research, the overwhelming complexity of cells has been a significant barrier to a fundamental understanding of how cells work as a whole. As such, the holistic study of biochemical pathways requires computer modeling. Due to the complexity of cells, it is not feasible for one person or group to model the cell in its entirety.

Results: The Cell Collective is a platform that allows the world-wide scientific community to create these models collectively. Its interface enables users to build and use models without specifying any mathematical equations or computer code - addressing …

Small But Crucial: The Novel Small Heat Shock Protein Hsp21 Mediates Stress Adaptation And Virulence In Candida Albicans, Francois L. Mayer, Duncan Wilson, Ilse D. Jacobsen, Pedro Miramón, Silvia Slesiona, Iryna Bohovych, Alistair J.P. Brown, Bernhard Hube

Department of Biochemistry: Faculty Publications

Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21∆/∆ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filamentinducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells …

Cellular Responses Of Candida Albicans To Phagocytosis And The Extracellular Activities Of Neutrophils Are Critical To Counteract Carbohydrate Starvation, Oxidative And Nitrosative Stress, Pedro Miramón, Christine Dunker, Hanna Windecker, Iryna Bohovych, Alistair J.P. Brown, Oliver Kurzai, Bernhard Hube

Department of Biochemistry: Faculty Publications

Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by upregulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in …

Role Of Apoptosis-Inducing Factor, Proline Dehydrogenase, And Nadph Oxidase In Apoptosis And Oxidative Stress, Sathish Kumar Natarajan, Donald F. Becker

Department of Biochemistry: Faculty Publications

Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF), proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In …

Substrate Channeling In Proline Metabolism, Benjamin W. Arentson, Nikhilesh Sanyal, Donald F. Becker

Department of Biochemistry: Faculty Publications

Proline metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. Results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism. One intermediate is pyrroline-5-carboxylate (P5C), which upon hydrolysis opens to glutamic semialdehyde (GSA). Recent structural and kinetic evidence indicate substrate channeling of P5C/ GSA occurs in the proline catabolic pathway between the proline dehydrogenase and P5C dehydrogenase active sites of bifunctional proline utilization A (PutA). Substrate channeling in PutA is proposed to facilitate the hydrolysis of P5C to GSA which is …

Late-Stage Maturation Of The Rieske Fe/S Protein: Mzm1 Stabilizes Rip1 But Does Not Facilitate Its Translocation By The Aaa Atpase Bcs1, Tie-Zhong Cui, Pamela M. Smith, Jennifer L. Fox, Oleh Khalimonchuk, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The final step in the assembly of the ubiquinol-cytochrome c reductase or bc₁ complex involves the insertion of the Rieske Fe/S cluster protein, Rip1. Maturation of Rip1 occurs within the mitochondrial matrix prior to its translocation across the inner membrane (IM) in a process mediated by the Bcs1 ATPase and subsequent insertion into the bc₁ complex. Here we show that the matrix protein Mzm1 functions as a Rip1 chaperone, stabilizing Rip1 prior to the translocation step. In the absence of Mzm1, Rip1 is prone to either proteolytic degradation or temperature-induced aggregation. A series of Rip1 truncations were engineered …

Wall_1.6 — Acknowledgements, Allison Marsh

Section 1: Introduction

No abstract provided.

Selective Oma1 Protease-Mediated Proteolysis Of Cox1 Subunit Of Cytochrome Oxidase In Assembly Mutants, Oleh Khalimonchuk, Mi-Young Jeong, Talina Watts, Elliott Ferris, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Background: Yeast lacking Coa2 are deficient in cytochrome c oxidase due to Cox1 degradation.

Results: Oma1 mediates Cox1 degradation in coa2∆ cells but not other mutants stalled in oxidase biogenesis.

Conclusion: Impaired hemylation of Cox1 in coa2∆ cells leads to misfolding and facile degradation by Oma1.

Significance: Oma1 functions in quality control of cytochrome oxidase assembly.

The Evolutionary Rewiring Of Ubiquitination Targets Has Reprogrammed The Regulation Of Carbon Assimilation In The Pathogenic Yeast Candida Albicans, Doblin Sandai, Zhikang Yin, Laura Selway, David Stead, Janet Walker, Michelle D. Leach, Iryna Bohovych, Iuliana V. Ene, Stavroula Kastora, Susan Budge, Carol A. Munro, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous …

Proline Dehydrogenase Is Essential For Proline Protection Against Hydrogen Peroxide Induced Cell Death, Sathish Kumar Natarajan, Weidong Zhu, Xinwen Liang, Lu Zhang, Andrew Demers, Matthew C. Zimmerman, Melanie A. Simpson, Donald F. Becker

Department of Biochemistry: Faculty Publications

Proline metabolism has an underlying role in apoptotic signaling that impacts tumorigenesis. Proline is oxidized to glutamate in the mitochondria with the rate limiting step catalyzed by proline dehydrogenase (PRODH). PRODH expression is inducible by p53 leading to increased proline oxidation, reactive oxygen species (ROS) formation, and induction of apoptosis. Paradoxical to its role in apoptosis, proline also protects cells against oxidative stress. Here we explore the mechanism of proline protection against hydrogen peroxide stress in melanoma WM35 cells. Treatment of WM35 cells with proline significantly increased cell viability, diminished oxidative damage of cellular lipids and proteins, and retained ATP …

Ergodic Sets As Cell Phenotype Of Budding Yeast Cell Cycle, Robert G. Todd, Tomáš Helikar

Department of Biochemistry: Faculty Publications

It has been suggested that irreducible sets of states in Probabilistic Boolean Networks correspond to cellular phenotype. In this study, we identify such sets of states for each phase of the budding yeast cell cycle. We find that these ‘‘ergodic sets’’ underly the cyclin activity levels during each phase of the cell cycle. Our results compare to the observations made in several laboratory experiments as well as the results of differential equation models. Dynamical studies of this model: (i) indicate that under stochastic external signals the continuous oscillating waves of cyclin activity and the opposing waves of CKIs …

Genome, Functional Gene Annotation, And Nuclear Transformation Of The Heterokont Oleaginous Alga Nannochloropsis Oceanica Ccmp1779, Astrid Vieler, Guangxi Wu, Chia-Hong Tsai, Blair Bullard, Adam J. Cornish, Christopher Harvey, Ida-Barbara Reca, Chelsea Thornburg, Rujira Achawanantakun, Christopher J. Buehl, Michael S. Campbell, David Cavalier, Kevin L. Childs, Teresa J. Clark, Rahul Deshpande, Erika Erickson, Ann Armenia Ferguson, Witawas Handee, Que Kong, Xiaobo Li, Bensheng Liu, Steven Lundback, Cheng Peng, Rebecca Roston, Michigan State University, Jeffrey P. Simpson, Allan Terbush, Jaruswan Warakanont, Simone Zäuner, Eva M. Farre, Eric L. Hegg, Ning Jiang, Min-Hao Kuo, Yan Lu, Krishna K. Niyogi, John Ohlrogge, Katherine W. Osteryoung, Yair Shachar-Hill, Barbara B. Sears, Yanni Sun, Hideki Takahashi, Mark Yandell, Shin-Han Shiu, Christoph Benning

Department of Biochemistry: Faculty Publications

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the …

Influence Of Peptide Dipoles And Hydrogen Bonds On Reactive Cysteine PkA Values In Fission Yeast Dj-1, Peter Madzelan, Tetyana Labunksa, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Cysteine residues with depressed pK_a values are critical for the functions of many proteins. Several types of interactions can stabilize cysteine thiolate anions, including hydrogen bonds between thiol(ate)s and nearby residues as well as electrostatic interactions involving charged residues or dipoles. Dipolar stabilization of thiolates by peptide groups has been suggested to play a particularly important role near the N-termini of α-helices. Using a combination of X-ray crystallography, site-directed mutagenesis, and spectroscopic methods, we show that the reactive cysteine residue (Cys111) in Schizosaccharomyces pombe DJ-1 experiences a 0.6 unit depression of its thiol pK_a as a consequence of …

A Framework Genetic Map For Miscanthus Sinensis From Rnaseq-Based Markers Shows Recent Tetraploidy, Kankshita Swaminathan, Won Byoung Chae, Therese Mitros, Kranthi Varala, Liang Xie, Adam Barling, Katarzyna Glowacka, Megan Hall, Stanislaw Jezowski, Ray Ming, Matthew Hudson, John A. Juvik, Daniel S. Rokhsar, Stephen P. Moose

Department of Biochemistry: Faculty Publications

Background: Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses whose high biomass production makes it, along with its close relatives sugarcane and sorghum, attractive as a biofuel feedstock. The base chromosome number of Miscanthus (x = 19) is different from that of other Saccharinae and approximately twice that of the related Sorghum bicolor (x = 10), suggesting largescale duplications may have occurred in recent ancestors of Miscanthus. Owing to the complexity of the Miscanthus genome and the complications of self-incompatibility, a complete genetic map with a high density of markers has not …

Structure, Function, And Assembly Of Heme Centers In Mitochondrial Respiratory Complexes, Hyung J. Kim, Oleh Khalimonchuk, Pamela M. Smith, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The sequential flow of electrons in the respiratory chain, from a low reduction potential substrate to O₂, is mediated by protein-bound redox cofactors. In mitochondria, hemes—together with flavin, iron–sulfur, and copper cofactors—mediate this multi-electron transfer. Hemes, in three different forms, are used as a protein-bound prosthetic group in succinate dehydrogenase (complex II), in bc₁ complex (complex III) and in cytochrome c oxidase (complex IV). The exact function of heme b in complex II is still unclear, and lags behind in operational detail that is available for the hemes of complex III and IV. The two b hemes …

Modeling An Improvement In Phosphorus Utilization In Tropical Agriculture, David Edelstein, David J. Tonjes

Technology & Society Faculty Publications

Studies of Terra Preta soils have generated interest in recreating their fertility elsewhere. Much of the research has focused on soil amendment charcoal (“biochar”). Terra Preta also contains bone fragments, producing a high concentration of phosphorus. Some forecast worldwide declines in phosphorus supplies, and better agricultural system management is required to improve phosphorus use efficiency. A conceptual model is offered to consider the influence of charcoal on bioavailability of phosphorus. The model describes a system where improvements in the chemical and biological condition of the soil result in increased phosphorus availability and cycling. Mechanisms of phosphorus/charcoal interaction are considered, and …

Bio-Logic Builder: A Non-Technical Tool For Building Dynamical, Qualitative Models, Tomáš Helikar, Bryan Kowal, Alex Madrahimov, Manish Shrestha, Jay Pedersen, Kahani Limbu, Ishwor Thapa, Thaine Rowley, Rahul Satalkar, Naomi Kochi, John Konvalina, Jim A. Rogers

Department of Biochemistry: Faculty Publications

Computational modeling of biological processes is a promising tool in biomedical research. While a large part of its potential lies in the ability to integrate it with laboratory research, modeling currently generally requires a high degree of training in mathematics and/or computer science. To help address this issue, we have developed a web-based tool, Bio- Logic Builder, that enables laboratory scientists to define mathematical representations (based on a discrete formalism) of biological regulatory mechanisms in a modular and non-technical fashion. As part of the user interface, generalized ‘‘biologic’’ modules have been defined to provide users with the building blocks for …