Biochemistry, Biophysics, and Structural Biology Commons^™

Transcriptomic And Proteomic Dynamics In The Metabolism Of A Diazotrophic Cyanobacterium, Cyanothece Sp. Pcc 7822 During A Diurnal Light-Dark Cycle, Louis Sherman

Louis A Sherman

Background: Cyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light-dark cycle. Utilizing transcriptomic and proteomic methods, we were able to quantify the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions.

Results: By combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four …

Effect Of Intracellular Expression Of Antimicrobial Peptide Ll-37 On Growth Of Escherichia Coli Strain Top10 Under Aerobic And Anaerobic Conditions, Wei Liu, Shi Dong, Fei Xu, Xue Wang, T. Withers, Hongwei Yu, Xin Wang

Hongwei Yu

Antimicrobial peptides (AMPs) can cause lysis of target bacteria by directly inserting themselves into the lipid bilayer. This killing mechanism confounds the identification of the intracellular targets of AMPs. To circumvent this, we used a shuttle vector containing the inducible expression of a human cathelicidin-related AMP, LL-37, to examine its effect on Escherichia coli TOP10 under aerobic and anaerobic growth conditions. Induction of LL-37 caused growth inhibition and alteration in cell morphology to a filamentous phenotype. Further examination of the E. coli cell division protein FtsZ revealed that LL-37 did not interact with FtsZ. Moreover, intracellular expression of LL-37 results …

Synchronous Opening And Closing Motions Are Essential For Camp-Dependent Protein Kinase A Signaling, Atul K. Srivastava, Leanna R. Mcdonald, Alessandro Cembran, Jonggul Kim, Larry R. Masterson, Christopher L. Mcclendon, Susan S. Taylor, Gianluigi Veglia

Larry Masterson

Variability Of Vitamins B1, B2 And Minerals Content In Baobab (Adansonia Digitata) Leaves In East And West Africa, Hyacinthe Traore, Charles Parkouda, Adama Korbo, Diarra Compaoré/Sérémé, Mamoudou H. Dicko Prof., Jan J. Svejgaard, Bréhima Diawara

Pr. Mamoudou H. DICKO, PhD

The regional variability and age-age correlation on vitamin B1, vitamin B2 and minerals (Ca, Mg, P, K, Cu, Fe, Mn, Na, and Zn) concentration in baobab leaves were investigated. Baobab was cultivated from seeds from 11 african countries. Vitamins B1 and B2 content were assessed using microbiological VitaFast kits methods and minerals by atomic absorption and flame spectrometry methods. Overall, the results showed a higher content of vitamin B2 compared to vitamin B1 with the highest vitamin B2 content (1.04 +- 0.05 mg/100 g DM) from Senegal. The highest iron (Fe) content of 26.39 mg/100 g was found in baobab …

Nuclear Transport Of Single Molecules: Dwell Times At The Nuclear Pore Complex, Ulrich Kubitscheck, David Grunwald, Andreas Hoekstra, Daniel Rohleder, Thorsten Kues, Jan Peter Siebrasse, Reiner Peters

David Grünwald

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of …

Intranuclear Binding Kinetics And Mobility Of Single Native U1 Snrnp Particles In Living Cells, David Grunwald, Beatrice Spottke, Volker Buschmann, Ulrich Kubitscheck

David Grünwald

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay …

Conversion Of Red Fluorescent Protein Into A Bright Blue Probe, Oksana M. Subach, Illia S. Gundorov, Masami Yoshimura, Fedor V. Subach, Jinghang Zhang, David Grunwald, Ekaterina A. Souslova, Dmitriy M. Chudakov, Vladislav V. Verkhusha

David Grünwald

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins …

Autonomy And Robustness Of Translocation Through The Nuclear Pore Complex: A Single-Molecule Study, Thomas Dange, David Grunwald, Antje Grunwald, Reiner Peters, Ulrich Kubitscheck

David Grünwald

All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapalpha2, kapbeta1, kapbeta1DeltaN44, and kapbeta2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy …

Nuclear Pore Component Nup98 Is A Potential Tumor Suppressor And Regulates Posttranscriptional Expression Of Select P53 Target Genes, Stephan Singer, Ruiying Zhao, Anthony M. Barsotti, Anette Ouwehand, Mina Fazollahi, Elias Coutavas, Kai Breuhahn, Olaf Neumann, Thomas Longerich, Tobias Pusterla, Maureen A. Powers, Keith M. Giles, Peter J. Leedman, Jochen Hess, David Grunwald, Harmen J. Bussemaker, Robert H. Singer, Peter Schirmacher, Carol Prives

David Grünwald

The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3'UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3sigma) to be similarly regulated by Nup98. The expression …

Nanofluidity Of Fatty Acid Hydrocarbon Chains As Monitored By Benchtop Time-Domain Nuclear Magnetic Resonance, David P. Cistola

David P Cistola

The functional properties of lipid-rich assemblies such as serum lipoproteins, cell membranes, and intracellular lipid droplets are modulated by the fluidity of the hydrocarbon chain environment. Existing methods for monitoring hydrocarbon chain fluidity include fluorescence, electron spin resonance, and nuclear magnetic resonance (NMR) spectroscopy; each possesses advantages and limitations. Here we introduce a new approach based on benchtop time-domain 1H NMR relaxometry (TD-NMR). Unlike conventional NMR spectroscopy, TD-NMR does not rely on the chemical shift resolution made possible by homogeneous, high-field magnets and Fourier transforms. Rather, it focuses on a multiexponential analysis of the time-decay signal. In this study, we …

Differential Muscle Hypertrophy Is Associated With Satellite Cell Numbers And Akt Pathway Activation Following Activin Type Iib Receptor Inhibition In Mtm1 P.R69c Mice, Michael Lawlor, Marissa Viola, Hui Meng, Rachel Edelstein, Fujun Liu, Ke Yan, Elizabeth Luna, Alexandra Lerch-Gaggl, Raymond Hoffmann, Christopher Pierson, Anna Buj-Bello, Jennifer Lachey, Scott Pearsall, Lin Yang, Cecilia Hillard, Alan Beggs

Elizabeth J. Luna

X-linked myotubular myopathy is a congenital myopathy caused by deficiency of myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-linked myotubular myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence …

Phytoconstituents Analysis, Antioxidant Capacity And Antimicrobial Properties Of Extracts From Laggera Aurita L. (Asteraceae), Crépin I Dibala, Kiessoun Konaté, Mamounata Diao, Maurice Ouédraogo, Mamoudou H. Dicko Prof.

Pr. Mamoudou H. DICKO, PhD

Objective: This research was designed to evaluate the polyphenolic content, antibacterial potency and antioxidant activity of extract and fractions from Laggera aurita L. (Asteraceae), a medicinal herbaceous from Burkina Faso. Methods: Folin ciocalteu and AlCl3 methods respectively were used for polyphenol contents. The antioxidant activities of the samples were evaluated by various in vitro assays like ferrous reducing antioxidant assay (FRAP), Total reducing power, 2, 2’-diphenyl-1-picrylhydrazyl (DPPH) scavenging and ABTS radical cation decolorization assays. In vitro antibacterial capacity of bioactive fractions were investigated by agar disc diffusion, micro-well dilution (MIC), Minimum Bactericidal Concentration (MBC) assay and at least the effect …

Development And Evaluation Of A Replicon Particle Vaccine Expressing The E2 Glycoprotein Of Bovine Viral Diarrhea Virus (Bvdv) In Cattle, John Dustin Loy, Jill Gander, Mark Mogler, Ryan Vander Veen, Julia Ridpath, Delbert Hank Harris, Kurt Kamrud

John Loy

Background: Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen. Findings: Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and …

Heterogeneous Rotational Diffusion Of A Fluorescent Probe In Lipid Monolayers, Christina M. Othon

Christina M Othon

The rotational correlation time of the lipid probe 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC) is measured using fluorescence anisotropy for two lipid species. We measure the rotational diffusion in a monolayer of 1,2-Didecanoyl-sn-glycero-3-phosphocholine (DPPC) which displays a phase transition at room temperature from the liquid expanded to the liquid-condensed phase. The constant rotational diffusion of the probe throughout the phase transition reflects the measurement of dynamics in only the liquid-expanded phase. We contrast the dynamic changes during this phase coexistence to the continuous density increase observed in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at room temperature. We observe a non-exponential decay of the probe diffusion consistent with heterogeneity …

Circulating Microrna Profiles In Human Patients With Acetaminophen Hepatotoxicity Or Ischemic Hepatitis, Jeanine Ward, Chitra Kanchagar, Isana Veksler-Lublinsky, Rosalind C. Lee, Mitchell R. Mcgill, Hartmut Jaeschke, Steven C. Curry, Victor R. Ambros

Victor R. Ambros

We have identified, by quantitative real-time PCR, hundreds of miRNAs that are dramatically elevated in the plasma or serum of acetaminophen (APAP) overdose patients. Most of these circulating microRNAs decrease toward normal levels during treatment with N-acetyl cysteine (NAC). We identified a set of 11 miRNAs whose profiles and dynamics in the circulation during NAC treatment can discriminate APAP hepatotoxicity from ischemic hepatitis. The elevation of certain miRNAs can precede the dramatic rise in the standard biomarker, alanine aminotransferase (ALT), and these miRNAs also respond more rapidly than ALT to successful treatment. Our results suggest that miRNAs can serve as …

The Chevrolet Cruze Luv 1.4 Engine, Gabriel Leiner

Gabriel Leiner

In the future, this research suggests that designing highways and cars with features built into the structures of the roads themselves that implicitly influence typical drivers to achieve better fuel economy without making an active effort. These types of “intuitively” fuel efficient highways and cars are proposed, defined and modeled within the scope of this paper.

Characterization Of A Recently Purified Thermophilic Dnase From A Novel Thermophilic Fungus, Kyle Landry, Robert Levin

Kyle S Landry

The Demethylating Agent 5-Aza Reduces The Growth, Invasiveness, And Clonogenicity Of Uveal And Cutaneous Melanoma, F. Rajaii, L. Asnaghi, Raymond Enke, S. Merbs, J. Handa, C. Eberhart

Ray Enke Ph.D.

No abstract provided.

Purification And Characterization Of Sweet Potato (Ipomoea Batatas) Peroxidase, Mamouna Diao, Brice N’Cho Ayékoué, Crépin I. Dibala, Soumaïla Dabonné, Mamoudou H. Dicko Prof.

Pr. Mamoudou H. DICKO, PhD

Peroxidase (POX), a biotechnologically important enzyme was purified from bulb of Ipomoea batatas and characterized for use in the bioconversion of phenolic compounds from industrial wastewater. Purified peroxidase (POXp) was obtained using the gel-filtration chromatography and anion exchange chromatography. A final yield of 18 % was obtained with 20.5 as purification fold. Electrophoresis on SDS-PAGE and native-PAGE showed that POXp is monomeric with a molecular weight of 44 kDa. The optimum pH and optimum temperature were respectively 6.0 and 45 ° C. All enzyme activity was retained for 2 hours at pH values between 4.0 to 9.0. While the enzyme …

An Improved Predictive Recognition Model For Cys2-His2 Zinc Finger Proteins, Ankit Gupta, Ryan G. Christensen, Heather A. Bell, Mathew Goodwin, Ronak Y. Patel, Manishi Pandey, Metewo Selase Enuameh, Amy L. Rayla, Cong Zhu, Stacey Thibodeau-Beganny, Michael H. Brodsky, J. Keith Joung, Scot A. Wolfe, Gary D. Stormo

Michael Brodsky

Cys2-His2 zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers. Given the large number of unique zinc fingers and assemblies present across eukaryotes, a comprehensive predictive recognition model that could accurately estimate the DNA-binding specificity of any ZFP based on …

Crystal Structure And Functional Assignment Of Yfau, A Metal Ion Dependent Class Ii Aldolase From Escherichia Coli K12, Dean Rea, Rebecca Hovington, John Rakus, John Gerlt, Vilmos Fu¨Lo¨P, Timothy Bugg, David Roper

John F. Rakus

One of the major challenges in the postgenomic era is the functional assignment of proteins using sequence- and structure-based predictive methods coupled with experimental validation. We have used these approaches to investigate the structure and function of theEscherichia coli K-12 protein YfaU, annotated as a putative 4-hydroxy-2-ketoheptane-1,7-dioate aldolase (HpcH) in the sequence databases. HpcH is the final enzyme in the degradation pathway of the aromatic compound homoprotocatechuate. We have determined the crystal structure of apo-YfaU and the Mg2+−pyruvate product complex. Despite greater sequence and structural similarity to HpcH, genomic context suggests YfaU is instead a 2-keto-3-deoxy sugar aldolase like the …

Truncation Of Type Iv Pilin Induces Mucoidy In Pseudomonas Aeruginosa Strain Pao579, T. Ryan Withers, F. Heath Damron, Yeshi Yin, Hongwei D. Yu

Hongwei Yu

Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host's defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ22). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the …

2-Acylamido Analogues Of N-Acetylglucosamine Prime Formation Of Chitin Oligosaccharides By Yeast Chitin Synthase 2, Jacob Gyore, Archana R. Parameswar, Carleigh F. F. Hebbard, Younghoon Oh, Erfei Bi, Alexei V. Demchenko, Neil P. Price, Peter Orlean

Alexei Demchenko

Molecular Engineering Of Side-Chain Liquid Crystalline Polymers By Living Polymerizations, Coleen Pugh, Alan L. Kiste

Coleen Pugh

“Living” anionic, cationic, metalloporphyrin and ring-opening metathesis polymerizations have been used to prepare well-defined side-chain liquid crystalline homopolymers, block and graft copolymers and statistical copolymers. This paper analyzes their successes and failures by reviewing the mechanistic aspects and experimental conditions of each type of polymerization, and identifies other classes of mesogenic monomers that could be polymerized in a controlled manner in the future. The emerging structure/property relationships are then identified using well-defined SCLCPs in which only one structural feature is varied while all others remain constant. The thermal transitions of liquid crystalline polymethacrylates, polynorbomenes and poly(viny1 ether)s reach their limiting …

New Multidetector Solution Could Lead To Safer Alternatives To Faulty Silicone Breast Implants, Judit D. Puskas, Geof Wyatt

Judit E. Puskas

The article discusses the effort of the University of Akron's Department of Chemical and Biomolecular Engineering to pursue research which aims to reduce or eliminate capsular contracture associated with breast implants. It notes that the new multidetector nanotechnology developed by researchers can be better alternative to silicone gel-filled breast implants. It mentions that the new technology can also help in early diagnosis and imaging of breast cancer.

Draft Genome Sequence Of A Stable Mucoid Strain Of Pseudomonas Aeruginosa Pao581 With A Muca25 Mutation, Yeshi Yin, T. Ryan Withers, John R. W. Govan, Shannon L. Johnson, Hongwei D. Yu

Hongwei Yu

A mutation in the mucA gene, which encodes a negative regulator of alginate production in Pseudomonas aeruginosa, is the main mechanism underlying the conversion to mucoidy in clinical isolates from patients with cystic fibrosis (CF). Here, we announce the draft genome sequence of the stable alginate-overproducing mucoid strain P. aeruginosa PAO581 with a mucA25 mutation, a derivative from the nonmucoid strains P. aeruginosa PAO381 and PAO1.

Evidence For Sigma Factor Competition In The Regulation Of Alginate Production By Pseudomonas Aeruginosa, Yeshi Yin, T. Ryan Withers, Xin Wang, Hongwei D. Yu

Hongwei Yu

Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU(AlgUA61V). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgUA61V, 2) an overexpression of the stringent starvation protein A …

Draft Genome Sequence Of A Mucoid Isolate Of Pseudomonas Aeruginosa Strain C7447m From A Patient With Cystic Fibrosis, Yeshi Yin, T. Ryan Withers, Shannon L. Johnson, Hongwei D. Yu

Hongwei Yu

Alginate overproduction by Pseudomonas aeruginosa, or mucoidy, plays an important role in the pathogenesis of chronic lung infections in cystic fibrosis (CF) patients. Here we report the draft genome sequence of a clinical isolate of mucoid P. aeruginosa strain C7447m from a CF patient with chronic lung infection.

Carbon Source-Dependent Alteration Of Puf3p Activity Mediates Rapid Changes In The Stabilities Of Mrnas Involved In Mitochondrial Function, Melanie A. Miller, Joseph Russo, Anthony D. Fischer, Florencia A. Lopez Leban, Wendy M. Olivas

Wendy Olivas

Acute Modulation Of Sugar Transport In Brain Capillary Endothelial Cell Cultures During Activation Of The Metabolic Stress Pathway, Anthony Cura, Anthony Carruthers

Anthony J. Cura

GLUT1-catalyzed equilibrative sugar transport across the mammalian blood-brain barrier is stimulated during acute and chronic metabolic stress; however, the mechanism of acute transport regulation is unknown. We have examined acute sugar transport regulation in the murine brain microvasculature endothelial cell line bEnd.3. Acute cellular metabolic stress was induced by glucose depletion, by potassium cyanide, or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which reduce or deplete intracellular ATP within 15 min. This results in a 1.7-7-fold increase in V(max) for zero-trans 3-O-methylglucose uptake (sugar uptake into sugar-free cells) and a 3-10-fold increase in V(max) for equilibrium exchange transport (intracellular [sugar] = extracellular [sugar]). …