Life Sciences Commons^™

An Ascorbate-Reducible Cytochrome B561 Is Localized In Macrophage Lysosomes, De-Liang Zhang, Dan Su, Alajos Berczi , Amy Vargas , Han H. Asard

Department of Biochemistry: Faculty Publications

Cytochromes b561 (Cyts b561) are a family of intrinsic membrane proteins involved in ascorbate-mediated transmembrane electron transport. The chromaffin granule Cyt b561 (CGCytb) is believed to transport electrons donated by extravesicular ascorbate (ASC) across the membrane to intravesicular monodehydroascorbate (MDA) supporting catecholamine synthesis in neuroendocrine tissues. Another isoform, the duodenal Cyt b561 (Dcytb), was reported to have ferric reductase activity, possibly facilitating intestinal iron uptake. Herein, a new Cyt b561 homologue, LCytb (for lysosomal Cytb561) was found expressed in the late endosomal–lysosomal membrane. LCytb shared high sequence similarity with CGCytb (45% identity) and Dcytb (42% identity). Moreover, four heme-coordinating His …

Hyaluronidase Expression Induces Prostate Tumor Metastasis In An Orthotopic Mouse Model, Joy L. Kovar, Mark A. Johnson, William M. Volcheck, Jiyan Chen, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factorconjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked …

Methods And Materials For Making And Using Transgenic Dicamba-Degrading Organisms, Donald P. Weeks, Xiao-Zhuo Wang, Patricia L. Herman

Department of Biochemistry: Faculty Publications

The invention provides isolated and at least partially-purified dicamba-degrading enzymes, isolated DNA molecules coding for dicamba-degrading enzymes, DNA constructs coding for dicamba-degrading enzymes, transgenic host cells comprising DNA coding for dicamba-degrading enzymes, and transgenic plants and plant parts comprising one or more cells comprising DNA coding for dicamba-degrading enzymes. Expression of the dicamba-degrading enzymes results in the production of dicamba-degrading organisms, including dicamba-tolerant plants. The invention further provides a method of controlling weeds in a field containing the transgenic dicamba-tolerant plants of the invention and a method of decontaminating a material containing dicamba comprising applying an effective amount of a …

Peroxidase-Dependent Apoplastic Oxidative Burst In Arabidopsis Required For Pathogen Resistance, Laurence V. Bindschedler, Julia Dewdney, Kris A. Blee, Julie M. Stone, Tsuneaki Asai, Julia Plotnikov, Carine Denoux, Tezni Hayes, Chris Gerrish, Dewi R. Davies, Frederick M. Ausubel, G. Paul Bolwell

Department of Biochemistry: Faculty Publications

The oxidative burst is an early response to pathogen attack leading to the production of reactive oxygen species (ROS) including hydrogen peroxide. Two major mechanisms involving either NADPH oxidases or peroxidases that may exist singly or in combination in different plant species have been proposed for the generation of ROS. We identified an Arabidopsis thaliana azide-sensitive but diphenylene iodonium-insensitive apoplastic oxidative burst that generates H₂O₂ in response to a Fusarium oxysporum cell-wall preparation. Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a type III peroxidase, French bean peroxidase type 1 (FBP1) exhibited an impaired oxidative burst and …

Concurrent Expression Of Hyaluronan Biosynthetic And Processing Enzymes Promotes Growth And Vascularization Of Prostate Tumors In Mice, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Aggressive cells in prostate cancer secrete extracellular hyaluronan (HA) as a result of up-regulated HA synthase enzymes HAS2 and HAS3. Combined detection of HA and the HA processing hyaluronidase enzyme Hyal1 in prostate tumors correlates with poor outcome. HA oligomers produced by hyaluronidases are potent angiogenic stimuli. We investigated the respective roles of HAS2 and Hyal1 using 22Rv1 human prostate tumor cells that lack both enzyme activities. Stable transfectants were selected for overexpression of Hyal1 or HAS2 and for coexpression of Hyal1 and HAS2. HAS2 overexpression elevated HA production and excess pericellular HA retention. However, HAS2-transfected tumor cell growth in …

S-Adenosylmethionine Stabilizes Cystathionine Β-Synthase And Modulates Redox Capacity, Anna Prudova, Zachary Bauman, Aaron Braun, Victor Vitvitsky, Shelly C. Lu, Ruma Banerjee

Department of Biochemistry: Faculty Publications

The transsulfuration pathway converts homocysteine to cysteine and represents the metabolic link between antioxidant and methylation metabolism. The first and committing step in this pathway is catalyzed by cystathionine β-synthase (CBS), which is subject to complex regulation, including allosteric activation by the methyl donor, S-adenosylmethionine (AdoMet). In this study, we demonstrate that methionine restriction leads to a >10-fold decrease in CBS protein levels, and pulse proteolysis studies reveal that binding of AdoMet stabilizes the protein against degradation by ≈12 kcal/mol. These observations predict that under pathological conditions where AdoMet levels are diminished, CBS, and therefore glutathione levels, will be reduced. …

Purification And Initial Biochemical Characterization Of Atp:Cob(I)Alamin Adenosyltransferase (Eutt) Enzyme Of Salmonella Enterica, Nicole R. Baun, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella

enterica was overproduced and enriched to ~70% homogeneity,

and its basic kinetic parameters were determined. Abundant

amounts of EutT protein were produced, but all of it remained

insoluble. Soluble active EutT protein (~70% homogeneous) was

obtained after treatment with detergent. Under conditions in which

cobalamin (Cbl) was saturating, _Km(ATP) = 10 µM, k_cat = 0.03 s^-1,

and V_max = 54.5 nM min^-1. Similarly, under conditions in which

MgATPwas saturating,K_m(Cbl) = 4.1µM, k_cat = 0.06 s^-1, andV_max= …

Sequential Processing Of A Mitochondrial Tandem Protein: Insights Into Protein Import In Schizosaccharomyces Pombe, Oleh Khalimonchuk, Martin Ott, Soledad Funes, Kai Ostermann, Gerhard Rödel, Johannes M. Herrmann

Department of Biochemistry: Faculty Publications

The sequencing of the genome of Schizosaccharomyces pombe revealed the presence of a number of genes encoding tandem proteins, some of which are mitochondrial components. One of these proteins (pre-Rsm22- Cox11) consists of a fusion of Rsm22, a component of the mitochondrial ribosome, and Cox11, a factor required for copper insertion into cytochrome oxidase. Since in Saccharomyces cerevisiae, Cox11 is physically attached to the mitochondrial ribosome, it was suggested that the tandem organization of Rsm22-Cox11 is used to covalently tie the mitochondrial ribosome to Cox11 in S. pombe. We report here that pre-Rsm22-Cox11 is matured in two subsequent …

A Characterization Of Subterranean Termites In Nebraska Using Micro-Morphological And Molecular Techniques, Timothy J. Husen, Shripat T. Kamble, Julie M. Stone

Department of Biochemistry: Faculty Publications

Recent studies have demonstrated a great potential for the use of molecular biology in Reticulitermes species identification and to study other aspects of termite biology. This study utilized micro-morphological identification and microsatellite Simple Sequence Length Polymorphic (SSLP) DNA genotyping to characterize subterranean termites collected from 73 locations across the state of Nebraska. Morphological characters of soldier termites from 30/73 collection sites revealed the presence of two species, Reticulitermes flavipes (Kollar) and R. tibialis Banks. Morphology of the gular plate ratio was used for species differentiation. Deoxyribonucelic acid (DNA) was extracted from termite workers (from all samples) and used as a …

Microsatellite Genotyping To Distinguish Colonies And Intraspecies Genetic Variation In The Eastern Subterranean Termite, Reticulitermes Flavipes (Isoptera: Rhinotermitidae), Timothy J. Husen, Shripat T. Kamble, Julie M. Stone

Department of Biochemistry: Faculty Publications

Microsatellite markers and fingerprinting techniques have demonstrated great potential for assessing colony affiliation in Reticulitermes species. This study utilized microsatellite allele fingerprints and examined variation at eight micro satellite loci within five different R. flavipes collection sites in each of Lancaster and Madison counties in Nebraska. In the termite populations from Lancaster County, five of the eight microsatellite loci (Rf 1-3, Rf 3-1, Rf 5-1 0, Rf 6-1, and Rf 24-2) provided informative genotypic variability allowing colony distinction. DNA sequence data at locus Rf24-2 also provided conclusive evidence that populations sampled were distinct colonies. Five micro satellite loci (Rf 3-1, …