Publications by authors named "Timothy Gallaher"

25 Publications

  • Page 1 of 1

The "evil tribe" spreads across the land: A dated molecular phylogeny provides insight into dispersal, expansion, and biogeographic relationships within one of the largest tribes of the sunflower family (Vernonieae: Compositae).

Am J Bot 2021 03 5;108(3):505-519. Epub 2021 Mar 5.

Bishop Museum, 1525 Bernice Street, Honolulu, Hawaii, 96817, USA.

Premise: With over 1500 species, the globally distributed Vernonieae is one of the most successful members of the largest family of flowering plants, the Compositae. However, due to its morphological complexity and limited geographic representation in previous studies, subtribal and biogeographic relationships are unclear. Here, new DNA sequence data spanning the geographic range of the tribe provides a taxonomically robust time-calibrated phylogeny, estimates migration pathways and timing of important biogeographic events, and allows inference of environmental factors that have contributed to the success of the Vernonieae worldwide.

Methods: Phylogenetic relationships were estimated for 368 taxa representing all Vernonieae subtribes. Molecular clock and ancestral range estimation analyses provide a framework for inference of the biogeographic history of the tribe.

Results: Relationships among the subtribes were established and correct placement determined for problematic taxa, along with the first model-based assessment of the biogeographic history of the tribe. The Vernonieae were estimated to have evolved ~50 mya. Africa was the first center of diversity, from which a single dispersal event established the monophyletic New World lineage. Long-distance dispersal from Africa and Brazil established the tribe on five continents and Oceania.

Conclusions: The New World lineage is monophyletic, but Old World taxa are not. New subtribal taxonomies are needed. Moquinieae are nested in Vernonieae. Long-distance dispersal from Africa beginning 45 mya was key to establishing the tribe's near-global distribution. Migration corridors created by volcanic mountain chains and iron-rich soils in Africa and the Americas promoted radiation and range expansion.
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http://dx.doi.org/10.1002/ajb2.1614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048643PMC
March 2021

Lineage-based functional types: characterising functional diversity to enhance the representation of ecological behaviour in Land Surface Models.

New Phytol 2020 10 31;228(1):15-23. Epub 2020 Jul 31.

Forest Ecosystems and Society, Oregon State University, Corvallis, OR, 97331, USA.

Process-based vegetation models attempt to represent the wide range of trait variation in biomes by grouping ecologically similar species into plant functional types (PFTs). This approach has been successful in representing many aspects of plant physiology and biophysics but struggles to capture biogeographic history and ecological dynamics that determine biome boundaries and plant distributions. Grass-dominated ecosystems are broadly distributed across all vegetated continents and harbour large functional diversity, yet most Land Surface Models (LSMs) summarise grasses into two generic PFTs based primarily on differences between temperate C grasses and (sub)tropical C grasses. Incorporation of species-level trait variation is an active area of research to enhance the ecological realism of PFTs, which form the basis for vegetation processes and dynamics in LSMs. Using reported measurements, we developed grass functional trait values (physiological, structural, biochemical, anatomical, phenological, and disturbance-related) of dominant lineages to improve LSM representations. Our method is fundamentally different from previous efforts, as it uses phylogenetic relatedness to create lineage-based functional types (LFTs), situated between species-level trait data and PFT-level abstractions, thus providing a realistic representation of functional diversity and opening the door to the development of new vegetation models.
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http://dx.doi.org/10.1111/nph.16773DOI Listing
October 2020

3D shape analysis of grass silica short cell phytoliths: a new method for fossil classification and analysis of shape evolution.

New Phytol 2020 10 1;228(1):376-392. Epub 2020 Jul 1.

University of Washington Biology Department, Life Sciences Building, Seattle, WA, 98105, USA.

Fossil grass silica short cell phytoliths (GSSCP) have been used to reconstruct the biogeography of Poaceae, untangle crop domestication history and detect past vegetation shifts. These inferences depend on accurately identifying the clade to which the fossils belong. Patterns of GSSCP shape and size variation across the family have not been established and current classification methods are subjective or based on a 2D view that ignores important 3D shape variation. Focusing on Poaceae subfamilies Anomochlooideae, Pharoideae, Pueliodieae, Bambusoideae and Oryzoideae, we observed in situ GSSCP to establish their orientation and imaged isolated GSSCP using confocal microscopy to produce 3D models. 3D geometric morphometrics was used to analyze GSSCP shape and size. Classification models were applied to GSSCP from Eocene sediments from Nebraska, USA, and Anatolia, Turkey. There were significant shape differences between nearly all recognized GSSCP morphotypes and between clades with shared morphotypes. Most of the Eocene GSSCP were classified as woody bamboos with some distinctive Nebraska GSSCP classified as herbaceous bamboos. 3D morphometrics hold great promise for GSSCP classification. It accounts for the complete GSSCP shape, automates size measurements and accommodates the complete range of morphotypes within a single analytical framework.
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http://dx.doi.org/10.1111/nph.16677DOI Listing
October 2020

Insights into the Evolutionary History of the Hawaiian Bidens (Asteraceae) Adaptive Radiation Revealed Through Phylogenomics.

J Hered 2020 02;111(1):119-137

USDA-ARS, Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center, Hilo, HI.

Hawaiian plant radiations often result in lineages with exceptionally high species richness and extreme morphological and ecological differentiation. However, they typically display low levels of genetic variation, hindering the use of classic DNA markers to resolve their evolutionary histories. Here we utilize a phylogenomic approach to generate the first generally well-resolved phylogenetic hypothesis for the evolution of the Hawaiian Bidens (Asteraceae) adaptive radiation, including refined initial colonization and divergence time estimates. We sequenced the chloroplast genome (plastome) and nuclear ribosomal complex for 18 of the 19 endemic species of Hawaiian Bidens and 4 outgroup species. Phylogenomic analyses based on the concatenated dataset (plastome and nuclear) resulted in identical Bayesian and Maximum Likelihood trees with high statistical support at most nodes. Estimates from dating analyses were similar across datasets, with the crown group emerging ~1.76-1.82 Mya. Biogeographic analyses based on the nuclear and concatenated datasets indicated that colonization within the Hawaiian Islands generally followed the progression rule with 67-80% of colonization events from older to younger islands, while only 53% of events followed the progression rule in the plastome analysis. We find strong evidence for nuclear-plastome conflict indicating a potentially important role for hybridization in the evolution of the group. However, incomplete lineage sorting cannot be ruled out due to the small number of independent loci analyzed. This study contributes new insights into species relationships and the biogeographic history of the explosive Hawaiian Bidens adaptive radiation.
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http://dx.doi.org/10.1093/jhered/esz066DOI Listing
February 2020

Leaf shape and size track habitat transitions across forest-grassland boundaries in the grass family (Poaceae).

Evolution 2019 05 26;73(5):927-946. Epub 2019 Mar 26.

Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, Iowa, 50011.

Grass leaf shape is a strong indicator of their habitat with linear leaves predominating in open areas and ovate leaves distinguishing forest-associated grasses. This pattern among extant species suggests that ancestral shifts between forest and open habitats may have coincided with changes in leaf shape or size. We tested relationships between habitat, climate, photosynthetic pathway, and leaf shape and size in a phylogenetic framework to evaluate drivers of leaf shape and size variation over the evolutionary history of the family. We also estimated the ancestral habitat of Poaceae and tested whether forest margins served as transitional zones for shifts between forests and grasslands. We found that grass leaf shape is converging toward different shape optima in the forest understory, forest margins, and open habitats. Leaf size also varies with habitat. Grasses have smaller leaves in open and drier areas, and in areas with high solar irradiance. Direct transitions between linear and ovate leaves are rare as are direct shifts between forest and open habitats. The most likely ancestral habitat of the family was the forest understory and forest margins along with an intermediate leaf shape served as important transitional habitat and morphology, respectively, for subsequent shifts across forest-grassland biome boundaries.
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http://dx.doi.org/10.1111/evo.13722DOI Listing
May 2019

A refined method for digitally modeling small and complex plant structures in 3D: An example from the grasses (Poaceae).

Appl Plant Sci 2018 Aug 27;6(8):e01177. Epub 2018 Aug 27.

Department of Ecology, Evolution, and Organismal Biology Iowa State University 2200 Osborn Drive Ames Iowa 50011 USA.

Premise Of The Study: A refined procedure is described for modeling small, intricate plant structures using computer-aided design software. The procedure facilitates the study of wind pollination in the family Poaceae and provides virtual biological illustrations for public outreach.

Methods And Results: Spikelets were fixed in gFAA, dehydrated using ethanol and xylene, embedded in paraffin wax, and then sectioned with a rotary microtome. Images of serial sections were used as a reference for modeling the shape of bracts with splines in a computer-aided design program. Virtual models produced by this method have many potential uses; examples include geometric morphometric analyses and simulations of computational fluid dynamics.

Conclusions: This protocol is a synthesis of modern biological illustration and engineering technology. Virtual models facilitate quantitative experiments that may address questions about reproductive biology, conditions shaping the form of anatomical support, or the morphological evolution of structures of biomechanical interest.
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http://dx.doi.org/10.1002/aps3.1177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110240PMC
August 2018

Topological Data Analysis as a Morphometric Method: Using Persistent Homology to Demarcate a Leaf Morphospace.

Front Plant Sci 2018 25;9:553. Epub 2018 Apr 25.

Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, United States.

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied as a filtration across simplicial complexes (or more simply, a method to measure topological features of spaces across different spatial resolutions), to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. The approach predicts plant family above chance. The application of a persistent homology method, using topological features, to measure leaf shape allows for a unified morphometric framework to measure plant form, including shapes, textures, patterns, and branching architectures.
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http://dx.doi.org/10.3389/fpls.2018.00553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996898PMC
April 2018

A long distance dispersal hypothesis for the Pandanaceae and the origins of the Pandanus tectorius complex.

Mol Phylogenet Evol 2015 Feb 13;83:20-32. Epub 2014 Nov 13.

University of Hawaii, Botany Department, 3190 Maile Way, Honolulu, HI 96822, USA.

Pandanaceae (screwpines) is a monocot family composed of c. 750 species widely distributed in the Paleotropics. It has been proposed that the family may have a Gondwanan origin with an extant Paleotropical distribution resulting from the breakup of that supercontinent. However, fossils supporting that hypothesis have been recently reassigned to other families while new fossil discoveries suggest an alternate hypothesis. In the present study, nuclear and chloroplast sequences were used to resolve relationships among Pandanaceae genera. Two well-supported fossils were used to produce a chronogram to infer whether the age of major intra-familial lineages corresponds with the breakup of Gondwana. The Pandanaceae has a Late Cretaceous origin, and genera on former Gondwanan landmasses began to diverge in the Late Eocene, well after many of the southern hemisphere continents became isolated. The results suggest an extant distribution influenced by long-distance-dispersal. The most widespread group within the family, the Pandanus tectorius species complex, originated in Eastern Queensland within the past six million years and has spread to encompass nearly the entire geographic extent of the family from Africa through Polynesia. The spread of that group is likely due to dispersal via hydrochory as well as a combination of traits such as agamospermy, anemophily, and multi-seeded propagules which can facilitate the establishment of new populations in remote locations.
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http://dx.doi.org/10.1016/j.ympev.2014.11.002DOI Listing
February 2015

Community impacts of Prosopis juliflora invasion: biogeographic and congeneric comparisons.

PLoS One 2012 12;7(9):e44966. Epub 2012 Sep 12.

Department of Environmental Studies, Centre for Environmental Management of Degraded Ecosystems (CEMDE), University of Delhi, Delhi, India.

We coordinated biogeographical comparisons of the impacts of an exotic invasive tree in its native and non-native ranges with a congeneric comparison in the non-native range. Prosopis juliflora is taxonomically complicated and with P. pallida forms the P. juliflora complex. Thus we sampled P. juliflora in its native Venezuela, and also located two field sites in Peru, the native range of Prosopis pallida. Canopies of Prosopis juliflora, a native of the New World but an invader in many other regions, had facilitative effects on the diversity of other species in its native Venezuela, and P. pallida had both negative and positive effects depending on the year, (overall neutral effects) in its native Peru. However, in India and Hawaii, USA, where P. juliflora is an aggressive invader, canopy effects were consistently and strongly negative on species richness. Prosopis cineraria, a native to India, had much weaker effects on species richness in India than P. juliflora. We carried out multiple congeneric comparisons between P. juliflora and P. cineraria, and found that soil from the rhizosphere of P. juliflora had higher extractable phosphorus, soluble salts and total phenolics than P. cineraria rhizosphere soils. Experimentally applied P. juliflora litter caused far greater mortality of native Indian species than litter from P. cineraria. Prosopis juliflora leaf leachate had neutral to negative effects on root growth of three common crop species of north-west India whereas P. cineraria leaf leachate had positive effects. Prosopis juliflora leaf leachate also had higher concentrations of total phenolics and L-tryptophan than P. cineraria, suggesting a potential allelopathic mechanism for the congeneric differences. Our results also suggest the possibility of regional evolutionary trajectories among competitors and that recent mixing of species from different trajectories has the potential to disrupt evolved interactions among native species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044966PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3440363PMC
March 2013

HemaMax™, a recombinant human interleukin-12, is a potent mitigator of acute radiation injury in mice and non-human primates.

PLoS One 2012 24;7(2):e30434. Epub 2012 Feb 24.

Neumedicines, Inc, Pasadena, California, United States of America.

HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8-9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit-expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD(50/30)) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12-14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030434PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286478PMC
August 2012

Modeling of the water network at protein-RNA interfaces.

J Chem Inf Model 2011 Jun 7;51(6):1347-52. Epub 2011 Jun 7.

Department of Pharmacology & Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90089-9121, United States.

Water plays an important role in the mediation of biomolecular interactions. Thus, accurate prediction and evaluation of water-mediated interactions is an important element in the computational design of interfaces involving proteins, RNA, and DNA. Here, we use an algorithm (WATGEN) to predict the locations of interfacial water molecules for a data set of 224 protein-RNA interfaces. The accuracy of the prediction is validated against water molecules present in the X-ray structures of 105 of these complexes. The complexity of the water networks is deconvoluted through definition of the characteristics of each water molecule based on its bridging properties between the protein and RNA and on its depth in the interface with respect to the bulk solvent. This approach has the potential for scoring the water network for incorporation into the computational design of protein-RNA complexes.
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http://dx.doi.org/10.1021/ci200118yDOI Listing
June 2011

hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33.

J Neurooncol 2011 Apr 31;102(2):197-211. Epub 2010 Jul 31.

Neumedicines Inc, 133 N Altadena Dr. #310, Pasadena, CA 91107, USA.

The completion of the Human Genome Project resulted in discovery of many unknown novel genes. This feat paved the way for the future development of novel therapeutics for the treatment of human disease based on novel biological functions and pathways. Towards this aim, we undertook a bioinformatics analysis of in-house microarray data derived from purified hematopoietic stem cell populations. This effort led to the discovery of HSS1 (Hematopoietic Signal peptide-containing Secreted 1) and its splice variant HSM1 (Hematopoietic Signal peptide-containing Membrane domain-containing 1). HSS1 gene is evolutionarily conserved across species, phyla and even kingdoms, including mammals, invertebrates and plants. Structural analysis showed no homology between HSS1 and known proteins or known protein domains, indicating that it was a truly novel protein. Interestingly, the human HSS1 (hHSS1) gene is located at chromosome 19q13.33, a genomic region implicated in various cancers, including malignant glioma. Stable expression of hHSS1 in glioma-derived A172 and U87 cell lines greatly reduced their proliferation rates compared to mock-transfected cells. hHSS1 expression significantly affected the malignant phenotype of U87 cells both in vitro and in vivo. Further, preliminary immunohistochemical analysis revealed an increase in hHSS1/HSM1 immunoreactivity in two out of four high-grade astrocytomas (glioblastoma multiforme, WHO IV) as compared to low expression in all four low-grade diffuse astrocytomas (WHO grade II). High-expression of hHSS1 in high-grade gliomas was further supported by microarray data, which indicated that mesenchymal subclass gliomas exclusively up-regulated hHSS1. Our data reveal that HSS1 is a truly novel protein defining a new class of secreted factors, and that it may have an important role in cancer, particularly glioma.
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http://dx.doi.org/10.1007/s11060-010-0314-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3052511PMC
April 2011

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

Genet Vaccines Ther 2009 Dec 30;7:13. Epub 2009 Dec 30.

Neumedicines Inc, Pasadena, California 91107, USA.

Background: Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.

Methods: A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.

Results: Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.

Conclusion: These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.
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http://dx.doi.org/10.1186/1479-0556-7-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2809042PMC
December 2009

Effects of age and calorie restriction on tryptophan nitration, protein content, and activity of succinyl-CoA:3-ketoacid CoA transferase in rat kidney mitochondria.

Free Radic Biol Med 2010 Feb 16;48(4):609-18. Epub 2009 Dec 16.

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033, USA.

This study examined the protein targets of nitration and the consequent impact on protein function in rat kidney mitochondria at 4, 13, 19, and 24 months of age. Succinyl-CoA transferase (SCOT), a rate-limiting enzyme in the degradation of ketone bodies, was the most intensely reactive protein against anti-3-nitrotyrosine antibody in rat kidney mitochondria. However, subsequent mass spectrometric and amino acid analyses of purified SCOT indicated that tryptophan 372, rather than a tyrosine residue, was the actual site of simultaneous additions of nitro and hydroxy groups. This finding suggests that identification of nitrated tyrosine residues based solely on reactivity with anti-3-nitrotyrosine antibody can be potentially misleading. Between 4 and 24 months of age, the amounts of SCOT protein and catalytic activity, expressed per milligram of mitochondrial proteins, decreased by 55 and 45%, respectively. SCOT, and particularly its nitrated carboxy-terminal region, was relatively more susceptible to in vitro proteolysis than other randomly selected kidney mitochondrial proteins. The age-related decreases in SCOT protein amount and catalytic activity were prevented by a relatively long-term 40% reduction in the amount of food intake. Loss of SCOT protein in the aged rats may attenuate the capacity of kidney mitochondria to utilize ketone bodies for energy production.
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http://dx.doi.org/10.1016/j.freeradbiomed.2009.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818783PMC
February 2010

Similarity of protein-RNA interfaces based on motif analysis.

J Chem Inf Model 2009 Sep;49(9):2139-46

Department of Pharmacology & Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90089-9121, USA.

We have developed a method for determination of the similarity of pairs of protein-RNA complexes, which we refer to as SIMA (Similarity by Identity and Motif Alignment). The key element in the SIMA method is the description of the protein-RNA interface in terms of motifs (salt bridges, aromatic stacking interactions, nonaromatic stacks, hydrophobic interactions, and hydrogen-bonded motifs), in addition to single hydrogen bonds and van der Waals contacts. Based on a pairwise scoring function combining motif alignment with identity of the protein and RNA sequences, we define a SIMA score for any pair of protein-RNA complexes. A positive score indicates similarity between the complexes. We used the SIMA method to identify 284 nonredundant binary protein-RNA complexes out of 776 such complexes in 382 nonribosomal protein-RNA structure files obtained from the RCSB database. SIMA allows rapid and quantitative comparison of protein-RNA interfaces and may be useful for interface classification with potential functional and evolutionary implications.
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http://dx.doi.org/10.1021/ci900154aDOI Listing
September 2009

Identification of IbeR as a stationary-phase regulator in meningitic Escherichia coli K1 that carries a loss-of-function mutation in rpoS.

J Biomed Biotechnol 2009 12;2009:520283. Epub 2009 Mar 12.

Saban Research Institute of Childrens Hospital Los Angeles, Department of Pediatrics, University of Southern California, Los Angeles, CA 90027, USA.

IbeR is a regulator present in meningitic Escherichia coli strain E44 that carries a loss-of-function mutation in the stationary-phase (SP) regulatory gene rpoS. In order to determine whether IbeR is an SP regulator in E44, two-dimensional gel electrophoresis and LC-MS were used to compare the proteomes of a noninvasive ibeR deletion mutant BR2 and its parent strain E44 in the SP. Four up-regulated (TufB, GapA, OmpA, AhpC) and three down-regulated (LpdA, TnaA, OpmC) proteins in BR2 were identified when compared to E44. All these proteins contribute to energy metabolism or stress resistance, which is related to SP regulation. One of the down-regulated proteins, tryptophanase (TnaA), which is regulated by RpoS in other E. coli strains, is associated with SP regulation via production of a signal molecule indole. Our studies demonstrated that TnaA was required for E44 invasion, and that indole was able to restore the noninvasive phenotype of the tnaA mutant. The production of indole was significantly reduced in BR2, indicating that ibeR is required for the indole production via tnaA. Survival studies under different stress conditions indicated that IbeR contributed to bacteria stress resistance in the SP. Taken together, IbeR is a novel regulator contributing to the SP regulation.
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http://dx.doi.org/10.1155/2009/520283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655632PMC
May 2009

LDL protein nitration: implication for LDL protein unfolding.

Arch Biochem Biophys 2008 Nov 7;479(1):1-14. Epub 2008 Aug 7.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.

Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL(-)) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL(-) assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC). The plasma LDL(-) fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL(-) revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the alpha-helical structures and beta(2) sheet as well as cysteine oxidation to cysteic acid in beta(1) sheet. Circular dichroism analyses showed that the alpha-helical content of LDL(-) was substantially lower ( approximately 25%) than that of native LDL ( approximately 90%); conversely, LDL(-) showed greater content of beta-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO(-)) or SIN-1: similar amino acid modifications as well as conformational changes (loss of alpha-helical structure and gain in beta-sheet structure) were observed. Both LDL(-) and ONOO(-)-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO(-)-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R. It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.
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http://dx.doi.org/10.1016/j.abb.2008.07.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649963PMC
November 2008

Detection and characterization of peroxynitrite-induced modifications of tyrosine, tryptophan, and methionine residues by tandem mass spectrometry.

Methods Enzymol 2008 ;441:283-94

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA.

Nitration and oxidation of tyrosine, tryptophan, and methionine residues in proteins are potential markers of their interaction with peroxynitrite. This chapter describes the procedure for the detection of these nitro-oxidative modifications by tandem mass spectrometry. The peptide YGDLANWMIPGK, shown to contain a nitrohydroxytryptophan in the mitochondrial enzyme succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT) in vivo, was synthesized and exposed to peroxynitrite in order to test whether an identical tryptophan derivative could be generated in vitro. Data show that the occurrence of specific fragmented ions corresponding to the oxidation of methionine, nitration of tyrosine, and nitration/oxidation of tryptophan residues can be used to identify the sites of the nitration and oxidation of proteins in vitro and in vivo. It is also demonstrated that a nitrohydroxy addition to the tryptophan, similar to that present in SCOT in vivo, can be produced in vitro.
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http://dx.doi.org/10.1016/S0076-6879(08)01215-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820399PMC
August 2008

Multilineage hematopoietic recovery with concomitant antitumor effects using low dose Interleukin-12 in myelosuppressed tumor-bearing mice.

J Transl Med 2008 May 19;6:26. Epub 2008 May 19.

Neumedicines Inc., 2275 East Foothill Blvd., Pasadena, California, USA.

Background: Interleukin-12 (IL-12) is a cytokine well known for its role in immunity. A lesser known function of IL-12 is its role in hematopoiesis. The promising data obtained in the preclinical models of antitumor immunotherapy raised hope that IL-12 could be a powerful therapeutic agent against cancer. However, excessive clinical toxicity, largely due to repeat dose regimens, and modest clinical response observed in the clinical trials have pointed to the necessity to design protocols that minimize toxicity without affecting the anti-tumor effect of IL-12. We have focused on the lesser known role of IL-12 in hematopoiesis and hypothesized that an important clinical role for IL-12 in cancer may be as an adjuvant hematological cancer therapy. In this putative clinical function, IL-12 is utilized for the prevention of cancer therapy-related cytopenias, while providing concomitant anti-tumor responses over and above responses observed with the primary therapy alone. This putative clinical function of IL-12 focuses on the dual role of IL-12 in hematopoiesis and immunity.

Methods: We assessed the ability of IL-12 to facilitate hematopoietic recovery from radiation (625 rad) and chemotherapy (cyclophosphamide) in two tumor-bearing murine models, namely the EL4 lymphoma and the Lewis lung cancer models. Antitumor effects and changes in bone marrow cellularity were also assessed.

Results: We show herein that carefully designed protocols, in mice, utilizing IL-12 as an adjuvant to radiation or chemotherapy yield facile and consistent, multilineage hematopoietic recovery from cancer therapy-induced cytopenias, as compared to vehicle and the clinically-utilized cytokine granulocyte colony-stimulating factor (G-CSF) (positive control), while still providing concomitant antitumor responses over and above the effects of the primary therapy alone. Moreover, our protocol design utilizes single, low doses of IL-12 that did not yield any apparent toxicity.

Conclusion: Our results portend that despite its past failure, IL-12 appears to have significant clinical potential as a hematological adjuvant cancer therapy.
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http://dx.doi.org/10.1186/1479-5876-6-26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2424034PMC
May 2008

Estradiol in vivo regulation of brain mitochondrial proteome.

J Neurosci 2007 Dec;27(51):14069-77

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, Pharmaceutical Sciences Center, and Program in Neuroscience, University of Southern California, Los Angeles, California 90033, USA.

We used a combined proteomic and functional biochemical approach to determine the overall impact of 17beta-estradiol (E2) on mitochondrial protein expression and function. To elucidate mitochondrial pathways activated by E2 in brain, two-dimensional (2D) gel electrophoresis was conducted to screen the mitoproteome. Ovariectomized adult female rats were treated with a single injection of E2. After 24 h of E2 exposure, mitochondria were purified from brain and 2D analysis and liquid chromatography-tandem mass spectrometry protein identification were conducted. Results of proteomic analyses indicated that of the 499 protein spots detected by image analysis, a total of 66 protein spots had a twofold or greater change in expression. Of these, 28 proteins were increased in expression after E2 treatment whereas 38 proteins were decreased in expression relative to control. E2 regulated key metabolic enzymes including pyruvate dehydrogenase, aconitase, and ATP-synthase. To confirm that E2-inducible changes in protein expression translated into functional consequences, we determined the impact of E2 on the enzymatic activity of the mitochondrial electron transport chain. In vivo, E2 treatment enhanced brain mitochondrial efficiency as evidenced by increased respiratory control ratio, elevated cytochrome-c oxidase activity and expression while simultaneously reducing free radical generation in brain. Results of these analyses provide insights into E2 mechanisms of regulating brain mitochondria, which have the potential for sustaining neurological health and prevention of neurodegenerative diseases associated with mitochondrial dysfunction such as Alzheimer's disease.
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http://dx.doi.org/10.1523/JNEUROSCI.4391-07.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673510PMC
December 2007

Nitration of tryptophan 372 in succinyl-CoA:3-ketoacid CoA transferase during aging in rat heart mitochondria.

Biochemistry 2007 Sep 8;46(35):10130-44. Epub 2007 Aug 8.

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90033, USA.

The main objective of this study was to test the hypothesis that in vivo post-translational modifications in proteins, induced by the endogenously generated reactive oxygen and nitrogen molecules, can alter protein function and thereby have an effect on metabolic pathways during the aging process. Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT), the mitochondrial enzyme involved in the breakdown of ketone bodies in the extrahepatic tissues, was identified in rat heart to undergo age-associated increase in a novel, nitro-hydroxy, addition to tryptophan 372, located in close proximity ( approximately 10 A) of the enzyme active site. Between 4 and 24 months of age, the molar content of nitration was more than doubled while specific enzyme activity increased significantly. The amount of SCOT protein, however, remained unchanged. In vitro treatment of heart mitochondrial soluble proteins with relatively low concentrations of peroxynitrite enhanced the nitration as well as specific activity of SCOT. Results of this study identify tryptophan to be a specific target of nitration in vivo, for the first time. We hypothesize that increases in tryptophan nitration of SCOT and catalytic activity constitute a plausible mechanism for the age-related metabolic shift toward enhanced ketone body consumption as an alternative source of energy supply in the heart.
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http://dx.doi.org/10.1021/bi7001482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526316PMC
September 2007

Forebrain-specific expression of monoamine oxidase A reduces neurotransmitter levels, restores the brain structure, and rescues aggressive behavior in monoamine oxidase A-deficient mice.

J Biol Chem 2007 Jan 7;282(1):115-23. Epub 2006 Nov 7.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089, USA.

Previous studies have established that abrogation of monoamine oxidase (MAO) A expression leads to a neurochemical, morphological, and behavioral specific phenotype with increased levels of serotonin (5-HT), norepinephrine, and dopamine, loss of barrel field structure in mouse somatosensory cortex, and an association with increased aggression in adults. Forebrain-specific MAO A transgenic mice were generated from MAO A knock-out (KO) mice by using the promoter of calcium-dependent kinase IIalpha (CaMKIIalpha). The presence of human MAO A transgene and its expression were verified by PCR of genomic DNA and reverse transcription-PCR of mRNA and Western blot, respectively. Significant MAO A catalytic activity, autoradiographic labeling of 5-HT, and immunocytochemistry of MAO A were found in the frontal cortex, striatum, and hippocampus but not in the cerebellum of the forebrain transgenic mice. Also, compared with MAO A KO mice, lower levels of 5-HT, norepinephrine, and DA and higher levels of MAO A metabolite 5-hydroxyindoleacetic acid were found in the forebrain regions but not in the cerebellum of the transgenic mice. These results suggest that MAO A is specifically expressed in the forebrain regions of transgenic mice. This forebrain-specific differential expression resulted in abrogation of the aggressive phenotype. Furthermore, the disorganization of the somatosensory cortex barrel field structure associated with MAO A KO mice was restored and became morphologically similar to wild type. Thus, the lack of MAO A in the forebrain of MAO A KO mice may underlie their phenotypes.
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http://dx.doi.org/10.1074/jbc.M609830200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2844870PMC
January 2007

Identification of biofilm proteins in non-typeable Haemophilus Influenzae.

BMC Microbiol 2006 Jul 19;6:65. Epub 2006 Jul 19.

Proteomic Core Facility, School of Pharmacy, Health Sciences Campus, University of Southern California, Los Angeles, CA 90033, USA.

Background: Non-typeable Haemophilus influenzae biofilm formation is implicated in a number of chronic infections including otitis media, sinusitis and bronchitis. Biofilm structure includes cells and secreted extracellular matrix that is "slimy" and believed to contribute to the antibiotic resistant properties of biofilm bacteria. Components of biofilm extracellular matrix are largely unknown. In order to identify such biofilm proteins an ex-vivo biofilm of a non-typeable Haemophilus influenzae isolate, originally from an otitis media patent, was produced by on-filter growth. Extracellular matrix fraction was subjected to proteomic analysis via LC-MS/MS to identify proteins.

Results: 265 proteins were identified in the extracellular matrix sample. The identified proteins were analyzed for COG grouping and predicted cellular location via the TMHMM and SignalP predictive algorithms. The most over-represented COG groups identified compared to their frequency in the Haemophilus influenzae genome were cell motility and secretion (group N) followed by ribosomal proteins of group J. A number of hypothetical or un-characterized proteins were observed, as well as proteins previously implicated in biofilm function.

Conclusion: This study represents an initial approach to identifying and cataloguing numerous proteins associated with biofilm structure. The approach can be applied to biofilms of other bacteria to look for commonalities of expression and obtained information on biofilm protein expression can be used in multidisciplinary approaches to further understand biofilm structure and function.
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http://dx.doi.org/10.1186/1471-2180-6-65DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1559630PMC
July 2006

Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione.

Biochemistry 2005 Sep;44(36):11986-96

Research Center for Liver Disease, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, California 90089-9121, USA.

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.
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http://dx.doi.org/10.1021/bi0509393DOI Listing
September 2005
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