Biochemistry, Biophysics, and Structural Biology Commons^™

The Interdomain Interface In Bifunctional Enzyme Protein 3/4a (Ns3/4a) Regulates Protease And Helicase Activities, Cihan Aydin, Sourav Mukherjee, Alicia Hanson, David Frick, Celia Schiffer

Celia A. Schiffer

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that …

Welcome To The Journal Of Evolution And Health, Aaron Blaisdell, Paul Jaminet, David C. Pendergrass

Aaron P Blaisdell

Welcome to the first issue of the Journal of Evolution and Health! The Journal of Evolution and Health is the peer-reviewed, open-access journal of the Ancestral Health Society, a community of scientists, healthcare professionals, and laypersons who collaborate to understand health challenges from an evolutionary perspective.

Characterizing The Promiscuity Of Ligab, A Lignin Catabolite Degrading Extradiol Dioxygenase From Sphingomonas Paucimobilis Syk-6, Kevin P. Barry, Erika A. Taylor

Erika A. Taylor, Ph.D.

LigAB from Sphingomonas paucimobilis SYK-6 is the only structurally characterized dioxygenase of the largely uncharacterized superfamily of Type II extradiol dioxygenases (EDO). This enzyme catalyzes the oxidative ring-opening of protocatechuate (3,4-dihydroxybenzoic acid or PCA) in a pathway allowing the degradation of lignin derived aromatic compounds (LDACs). LigAB has also been shown to utilize two other LDACs from the same metabolic pathway as substrates, gallate, and 3-O-methyl gallate; however, kcat/KM had not been reported for any of these compounds. In order to assess the catalytic efficiency and get insights into the observed promiscuity of this enzyme, steady-state kinetic analyses were performed …

Stomatin-Like Protein 2 Deficiency In T Cells Is Associated With Altered Mitochondrial Respiration And Defective Cd4+ T Cell Responses., Darah A Christie, Panagiotis Mitsopoulos, Julianna Blagih, Stanley D Dunn, Julie St-Pierre, Russell G Jones, Grant M Hatch, Joaquín Madrenas

Stanley D Dunn

Stomatin-like protein 2 (SLP-2) is a mostly mitochondrial protein that regulates mitochondrial biogenesis and function and modulates T cell activation. To determine the mechanism of action of SLP-2, we generated T cell-specific SLP-2-deficient mice. These mice had normal numbers of thymocytes and T cells in the periphery. However, conventional SLP-2-deficient T cells had a posttranscriptional defect in IL-2 production in response to TCR ligation, and this translated into reduced CD4(+) T cell responses. SLP-2 deficiency was associated with impaired cardiolipin compartmentalization in mitochondrial membranes, decreased levels of the NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, NADH dehydrogenase (ubiquinone) 1β subcomplex subunit …

Paracrine Action Of Transforming Growth Factor-Alpha In Rectal Crypt Epithelium Of Humans, Ivan Cameron, W. Hardman

Elaine Hardman Ph.D.

Colon and rectal mucosal crypt epithelium is a rapidly renewing cell population, where cell proliferation is normally balanced by cell loss. This report concerns the putative paracrine action of transforming growth factor α(TGF-α) in this homeostatic process. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and TGF-α was performed on biopsy specimens of rectal mucosa taken from consenting patients. The height of the proliferative compartment in mid-axially sectioned crypts in each individual was determined from the distribution of PCNA stained cells. The number of TGF-α stained cells that exhibited intense positive staining in a continuous column from the mouth down …

Comparative Metabolism Of Phenanthro[3,4-B]Thiophene And Benzo[C]Phenanthrene, Jaquan M. Williams

Jaquan M Williams

ABSTRACT OF THESIS Comparative Metabolism of Phenanthro[3,4-b]Thiophene And Benzo[c]Phenanthrene Polycyclic aromatic hydrocarbons (PAHs) and their sulfur-heterocyclic analogs (thia-PAHs) are commonly occurring persistent environmental contaminants formed by incomplete combustion of organic matter. A number of thia-PAHs have shown significant mutagenic and carcinogenic activities. As noted with PAHs, these chemical contaminants also require metabolic activation in order to exhibit their mutagenic and carcinogenic effects. In the present study, a comparison of the metabolism of highly mutagenic phenanthro[3,4-b]thiophene (P[3,4-b]T) and its weakly mutagenic carbon analogue, benzo[c]phenanthrene (B[c]P), was investigated. Metabolism studies were conducted using liver microsomes from induced rats, un-induced rats, as well …

Escherichia Coli Heptosyltransferase I: Investigation Of Protein Dynamics Of A Gt-B Structural Enzyme, Erika A. Taylor, Daniel J. Czyzyk, Shreya S. Sawant, Carlos A. Ramirez-Mondragon

Erika A. Taylor, Ph.D.

Heptosyltransferase I (HepI), the enzyme responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide, is a member of the GT-B structural class of enzymes. Crystal structures have revealed open and closed conformations of apo and ligand-bound GT-B enzymes, implying that large-scale protein conformational dynamics play a role in their reaction mechanism. Here we report transient kinetic analysis of conformational changes in HepI reported by intrinsic tryptophan fluorescence and present the first real-time evidence of a GT-B enzyme undergoing a substrate binding-induced transition from an open to closed state prior to catalysis.

Inwardly Rectifying Potassium Channels In Drosophila, Zhuo Luan, Hong-Sheng Li

zhuo luan

Inwardly rectifying potassium channels (Kir) are a special subset of potassium selective ion channels which pass potassium more easily into rather than out of the cell. These channels mediate a variety of cellular functions, including control of membrane resting potential, maintenance of potassium homeostasis and regulation of cellular metabolism. Given the existence of fifteen Kir genes in mammals, current genetic studies using mutant animals that lack a single channel may have missed many important physiological functions of these channels due to gene redundancy. This issue can be circumvented by using a simple model organism like Drosophila, whose genome encodes only …

Function And X-Ray Crystal Structure Of Escherichia Coli Yfde, Elwood A. Mullins, Kelly L. Sullivan, T. Joseph Kappock

T. Joseph Kappock

Many food plants accumulate oxalate, which humans absorb but do not metabolize, leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by converting it to oxalyl-CoA, which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl-CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria, including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT), YfdU (OXC), and YfdE, a class III CoA-transferase that is ~30% identical to YfdW. YfdW and YfdU are necessary …

Cooperative Effects Of Drug-Resistance Mutations In The Flap Region Of Hiv-1 Protease, Jennifer Foulkes-Murzycki, Christina Rosi, Nese Yilmaz, Robert Shafer, Celia Schiffer

Celia A. Schiffer

Understanding the interdependence of multiple mutations in conferring drug resistance is crucial to the development of novel and robust inhibitors. As HIV-1 protease continues to adapt and evade inhibitors while still maintaining the ability to specifically recognize and efficiently cleave its substrates, the problem of drug resistance has become more complicated. Under the selective pressure of therapy, correlated mutations accumulate throughout the enzyme to compromise inhibitor binding, but characterizing their energetic interdependency is not straightforward. A particular drug resistant variant (L10I/G48V/I54V/V82A) displays extreme entropy-enthalpy compensation relative to wild-type enzyme but a similar variant (L10I/G48V/I54A/V82A) does not. Individual mutations of sites …

Protein Translocation Across The Rough Endoplasmic Reticulum, Elisabet Mandon, Steven Trueman, Reid Gilmore

Elisabet Mandon

The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration.

Translocation Channel Gating Kinetics Balances Protein Translocation Efficiency With Signal Sequence Recognition Fidelity, Steven Trueman, Elisabet Mandon, Reid Gilmore

Elisabet Mandon

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence-binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in …

Structural And Thermodynamic Basis Of Amprenavir/Darunavir And Atazanavir Resistance In Hiv-1 Protease With Mutations At Residue 50, Seema Mittal, Rajintha Bandaranayake, Nancy King, Moses Prabu-Jeyabalan, Madhavi Nalam, Ellen Nalivaika, Nese Yilmaz, Celia Schiffer

Celia A. Schiffer

Drug resistance occurs through a series of subtle changes that maintain substrate recognition but no longer permit inhibitor binding. In HIV-1 protease, mutations at I50 are associated with such subtle changes that confer differential resistance to specific inhibitors. Residue I50 is located at the protease flap tips, closing the active site upon ligand binding. Under selective drug pressure, I50V/L substitutions emerge in patients, compromising drug susceptibility and leading to treatment failure. The I50V substitution is often associated with amprenavir (APV) and darunavir (DRV) resistance, while the I50L substitution is observed in patients failing atazanavir (ATV) therapy. To explain how APV, …

Crystal Structure Of The Dna Cytosine Deaminase Apobec3f: The Catalytically Active And Hiv-1 Vif-Binding Domain, Markus-Frederik Bohn, Shivender Shandilya, John Albin, Takahide Kouno, Brett Anderson, Rebecca Mcdougle, Michael Carpenter, Anurag Rathore, Leah Evans, Ahkillah Davis, Jingying Zhang, Yongjian Lu, Mohan Somasundaran, Hiroshi Matsuo, Reuben Harris, Celia Schiffer

Celia A. Schiffer

Human APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this …

A High Omega-3 Fatty Acid Diet Has Different Effects On Early And Late Stage Myeloid Progenitors, Melinda Varney, James Buchanan, Yulia Dementieva, W. Hardman, Vincent Sollars

Yulia Dementieva

The effects of the polyunsaturated omega-3 (n-3) and omega-6 (n-6) fatty acids (FA) on hematopoiesis are complex in that both FA forms are processed into leukotrienes, eicosanoids, and prostaglandins, which can have independent effects. These FA have antagonistic effects in that n-6 FA prostaglandins tend to be pro-proliferative and pro-inflammatory, while the effects of n-3 FA prostaglandins are the opposite. We have previously shown that diets high in n-3 FA reduce the size of the middle to later stage myeloid progenitor compartment in FVB X sv129 F1hybrid mice. To assay the effects of high n-3 FA diets on earlier stages …

Global Analysis Of Gene Expression Changes During Retinoic Acid-Induced Growth Arrest And Differentiation Of Melanoma: Comparison To Differentially Expressed Genes In Melanocytes Vs Melanoma, Mary H. Estler, Goran Boskovic, James Denvir, Sarah Miles, Donald A. Primerano, Richard M. Niles

Goran Boskovic

BACKGROUND: The incidence of malignant melanoma has significantly increased over the last decade. Some of these malignancies are susceptible to the growth inhibitory and pro-differentiating effects of all-trans-retinoic acid (RA). The molecular changes responsible for the biological activity of RA in melanoma are not well understood. RESULTS: In an analysis of sequential global gene expression changes during a 4-48 h RA treatment of B16 mouse melanoma cells, we found that RA increased the expression of 757 genes and decreased the expression of 737 genes. We also compared the gene expression profile (no RA treatment) between non-malignant melan-a mouse melanocytes and …

Human Monoclonal Antibody Mbl-Hcv1 Delays Hcv Viral Rebound Following Liver Transplantation: A Randomized Controlled Study, R. Chung, F. Gordon, M. Curry, T. Schiano, S. Emre, K. Corey, J. Markmann, M. Hertl, J. Pomposelli, E. Pomfret, S. Florman, M. Schilsky, Teresa Broering, Robert Finberg, Gyongyi Szabo, Phillip Zamore, U. Khettry, Gregory Babcock, Donna Ambrosino, Brett Leav, Mark Leney, H. Smith, Deborah Molrine

Gyongyi Szabo

Rapid allograft infection complicates liver transplantation (LT) in patients with hepatitis C virus (HCV). Pegylated interferon-alpha and ribavirin therapy after LT has significant toxicity and limited efficacy. The effect of a human monoclonal antibody targeting the HCV E2 glycoprotein (MBL-HCV1) on viral clearance was examined in a randomized, double-blind, placebo-controlled pilot study in patients infected with HCV genotype 1a undergoing LT. Subjects received 11 infusions of 50 mg/kg MBL-HCV1 (n=6) or placebo (n=5) intravenously with three infusions on day of transplant, a single infusion on days 1 through 7 and one infusion on day 14 after LT. MBL-HCV1 was well-tolerated …

Current State Of Food Security: Research, Education And Outreach In Burkina Universities, Mamoudou H. Dicko Prof.

Pr. Mamoudou H. DICKO, PhD

Burkina Faso is a Sahelian West African country. Current population is about 16 millions and 43.9 % live below the poverty line. Burkina Faso is classified as both a least developed country and a low-income, food-deficit country. The nutritional quality is poor (1830 against 2000 Kcal/person/day). Food insecurity affects 50 % of households, and is increasing in the urban areas. However, Burkina Faso is one of the country that respect the 2003 Maputo’s declaration’s call to invest at least 10 percent of the budget in agriculture, in order to increase agricultural production and attain food self-sufficiency. Several training programs dealing …

Assessing Tissue Characterization Of Abdominal Organs Using Fuzzy C-Means Cluster Analysis Of Color- Fusion Mr Images, Terrance D. Weeden, H. Keith Brown

Terrance Weeden

Genetic Parameters For Concentrations Of Minerals In Longissimus Muscle And Their Associations With Palatability Traits In Angus Cattle, R. G. Mateescu, A. J. Garmyn, Richard G. Tait Jr., Qing Duan, Q. Liu, Mary S. Mayes, Dorian J. Garrick, A. L. Van Eenennaam, D. L. Vanoverbeke, G. G. Hilton, Donald C. Beitz, James M. Reecy

Richard G. Tait Jr.

The objective of this study was to estimate genetic parameters for concentrations of minerals in LM and to evaluate their associations with beef palatability traits. Samples of LM from 2,285 Angus cattle were obtained and fabricated into steaks for analysis of mineral concentrations and for trained sensory panel assessments. Nine minerals, including calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc, were quantified. Restricted maximum likelihood procedures were used to obtain estimates of variance and covariance components under a multiple-trait animal model. Estimates of heritability for mineral concentrations in LM varied from 0.01 to 0.54. Iron and sodium were …

Genetic Parameters For Carnitine, Creatine, Creatinine, Carnosine, And Anserine Concentration In Longissimus Muscle And Their Association With Palatability Traits In Angus Cattle, R. G. Mateescu, A. J. Garmyn, M. A. O'Neil, Richard G. Tait Jr., Almass A. Abuzaid, Mary S. Mayes, Dorian J. Garrick, A. L. Van Eenennaam, D. L. Vanoverbeke, G. G. Hilton, Donald C. Beitz, James M. Reecy

Richard G. Tait Jr.

The objective of this study was to estimate genetic parameters for carnitine, creatine, creatinine, carnosine, and anserine concentration in LM and to evaluate their associations with Warner-Bratzler shear force (WBSF) and beef palatability traits. Longissimus muscle samples from 2,285 Angus cattle were obtained and fabricated into steaks for analysis of carnitine, creatine, creatinine, carnosine, anserine, and other nutrients, and for trained sensory panel and WBSF assessments. Restricted maximum likelihood procedures were used to obtain estimates of variance and covariance components under a multiple-trait animal model. Estimates of heritability for carnitine, creatine, creatinine, carnosine, and anserine concentrations in LM from Angus …

Tca Cycle Inactivation In Staphylococcus Aureus Alters Nitric Oxide Production In Raw 264.7 Cells, Chandirasegaran Massilamany, Arunakumar Gangaplara, Donald Gardner, James Musser, David Steffen, Greg Somerville, Jay Reddy

Greg A. Somerville

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts …

Acute Toxicity Of Copper Sulfate And Potassium Dichromate On Stygobiont Proasellus: General Aspects Of Groundwater Ecotoxicology And Future Perspectives, Ana Reboleira, Nelson Abrantes, Pedro Oromí, Fernando Gonçalves

Ana Sofia P.S. Reboleira

Karst systems harbor large groundwater resources for human consumption and represent an important habitat for rare and unprotected specialized animals, the so-called stygofauna. Due to the highly adapted features towards underground life, together with the geographic isolation provided by the subterranean aquifers, groundwater-dwelling animals may lose the ability to face sudden changes on their ecosystems, and therefore the risk of extinction is remarkably high. A little is known about their sensitiveness, especially linked to contamination pressure in urbanized karst areas. Understanding the impact of contaminants on stygofauna is important for setting groundwater environmental quality and management of karst systems. We …

Proximate Composition Of Traditional Local Sorghum Beer “ Dolo ” Manufactured In Ouagadougou, Fatouma Mohamed Abdoul-Latif, Mamoudou H. Dicko Prof.

Pr. Mamoudou H. DICKO, PhD

Dolo is a local beer manufactured from malted red sorghum grains. It is the most commonly consumed alcoholic beverage in Burkina Faso. Thirty samples of dolo, were collected from local markets in Ouagadougou and analyzed with respect to their proximate compositions and pH values. The average values of pH, dry matter and insoluble matter among samples were 3.5, 5.90 % and 0.85 %, respectively. Alcohol content of dolo samples was on average 2.3 % (v/v). The content of total proteins, carbohydrates and reducing sugars were 26, 38 and 10 µg/ml, respectively. Only traces of lipids were detected in all dolo …

The Intermediate Monoclinic Phase Of Phosphatidylcholines, Elizabeth Luna, Harden Mcconnell

Elizabeth J. Luna

Two pure phospholipids, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, have been studied using freeze-fracture electron microscopy and the partitioning of the spin label, TEMPO. It is found that the characteristic band pattern, corresponding to monoclinic symmetry in multilamellar liposomes, is observed only in freeze-fracture electron microphotographs when samples are quenched from temperatures intermediate between the chain melting transition temperature and the pretransition temperature of the membrane. Markings are also observed on fracture faces of samples quenched from below the pretransition, but these "bands" are few in number and are widely and irregularly spaced. The lipid membranes used for freeze-fracture were prepared …

Lateral Phase Separations In Binary Mixtures Of Phospholipids Having Different Charges And Different Crystalline Structures, Elizabeth Luna, Harden Mcconnell

Elizabeth J. Luna

Synthetic dipalmitoyl phosphatidylserine exhibits a sharp chain-melting transition temperature at 51 degrees C as judged by partitioning of the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl. Phase diagrams representing lateral phase separations in binary mixtures of dipalmitoyl phosphatidylserine with dipalmitoyl phosphatidylcholine as well as with dimyristoyl phosphatidylcholine are derived from paramagnetic resonance determinations of 2,2,6,6,-tetramethylpiperidine-1-oxyl partitioning, freeze-fracture electron microscopic studies and theoretical arguments that limit the general form of acceptable phase diagrams. The reported phase diagrams are the first to describe binary mixtures in which one lipid is charged and the second lipid uncharged. These phase diagrams also are the first to include the …

Multiple Phase Equilibria In Binary Mixtures Of Phospholipids, Elizabeth Luna, Harden Mcconnell

Elizabeth J. Luna

Approximate phse diagrams describing lateral phase separations are given for binary mixtures of dimyristoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine, distearoyl phosphatidycholine, and dipalmitoyl phosphatidylethanolamine. These diagrams are based in part on freeze-fracture electron microscopic data. These phase diagrams represent an improvement over previous studies in that both solid phses (Pbeta' and Lbeta') of the phosphatidylcholines are included. Further consideration is given to the problem of binary mixtures in which there are two Pbeta' phases that do not form a continuous range of solid solutions.

Characterization Of Myxococcus Xanthus Mazf And Implications For A New Point Of Regulation, Tye O. Boynton, Jonathan L. Mcmurry, Lawrence J. Shimkets

Jonathan McMurry

During development, Myxococcus xanthus cells undergo programmed cell death (PCD) whereby 80% of vegetative cells die. Previously, the MazF RNA interferase has been implicated in this role. Recently, it was shown that deletion of the mazF gene does not eliminate PCD in wild-type strain DK1622 as originally seen in DZF1. To clarify the role of MazF, recombinant enzyme was characterized using a highly sensitive assay in the presence and absence of the proposed antitoxin MrpC. In contrast to previous reports that MrpC inhibits MazF activity, the hydrolysis rate was enhanced in a concentration-dependent manner with MrpC or MrpC2, an N-terminally …

Transmembrane Domain Three Contributes To The Ion Conductance Pathway Of Channelrhodopsin-2, Robert Dempski, Olga Gaiko

Robert E. Dempski

Channelrhodopsin-2 (ChR2) is a light-activated nonselective cation channel that is found in the eyespot of the unicellular green alga Chlamydomonas reinhardtii. Despite the wide employment of this protein to control the membrane potential of excitable membranes, the molecular determinants that define the unique ion conductance properties of this protein are not well understood. To elucidate the cation permeability pathway of ion conductance, we performed cysteine scanning mutagenesis of transmembrane domain three followed by labeling with methanethiosulfonate derivatives. An analysis of our experimental results as modeled onto the crystal structure of the C1C2 chimera demonstrate that the ion permeation pathway includes …

Dna Damage Repair Genes Controlling Human Papillomavirus (Hpv) Episome Levels Under Conditions Of Stability And Extreme Instability, Terri G. Edwards, Thomas J. Vidmar, Kevin Koeller, James K. Bashkin, Chris Fisher

James Bashkin