Biochemistry, Biophysics, and Structural Biology Commons^™

The Wasp-Like Protein Scar Regulates Macropinocytosis, Phagocytosis And Endosomal Membrane Flow In Dictyostelium, David J. Seastone, Ed Harris, Lesly A. Temesvari, James E. Bear, Charles L. Saxe, James Cardelli

Department of Biochemistry: Faculty Publications

Scar, a member of the WASp protein family, was discovered in Dictyostelium discoideum during a genetic screen for second-site mutations that suppressed a developmental defect. Disruption of the scar gene reduced the levels of cellular F-actin by 50%. To investigate the role of Scar in endocytosis, phagocytosis and endocytic membrane trafficking, processes that depend on actin polymerization, we have analyzed a Dictyostelium cell line that is genetically null for Scar. Rates of fluid phase macropinocytosis and phagocytosis are significantly reduced in the scar^- cell-line. In addition, exocytosis of fluid phase is delayed in these cells and movement of fluid …

Rab11-Like Gtpase Associates With And Regulates The Structure And Function Of The Contractile Vacuole System In Dictyostelium, Ed Harris, Kunito Yoshida, James Cardelli, John Bush

Department of Biochemistry: Faculty Publications

Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80% identical at the amino acid level to mammalian Rab11. Subcellular fractionation and immunofluorescence studies revealed that DdRab11 was exclusively associated with the ATPase proton pump-rich contractile vacuole membrane system, consisting of a reticular network and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed that this Rab was associated with contractile vacuolar bladders undergoing formation, fusion and expulsion of water. The association of DdRab11 with contractile vacuole membranes was disrupted when cells were exposed to either hypo-osmotic conditions or an inhibitor of the ATPase …

Selenoprotein Oxidoreductase With Specificity For Thioredoxin And Glutathione Systems, Qi-An Sun, Leo Kirnarsky, Simon Sherman, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Thioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity …

The Cia5 Gene Controls Formation Of The Carbon Concentrating Mechanism In Chlamydomonas Reinhardtii, Youbin Xiang, Jun Zhang, Donald P. Weeks

Department of Biochemistry: Faculty Publications

Wild-type Chlamydomonas reinhardtii cells shifted from high concentrations (5%) of CO2 to low, ambient levels (0.03%) rapidly increase transcription of mRNAs from several CO₂-responsive genes. Simultaneously, they develop a functional carbon concentrating mechanism that allows the cells to greatly increase internal levels of CO₂ and HCO₃. The cia5 mutant is defective in all of these phenotypes. A newly isolated gene, designated Cia5, restores transformed cia5 cells to the phenotype of wild-type cells. The 6,481-bp gene produces a 5.1-kb mRNA that is present constitutively in light in high and low CO₂ both in …

C3 Origins Of The C4 Pathway Regulatory Enzyme, Ppdk-Rp, C. J. Chastain, Gautam Sarath, Raymond Chollet

Gautam Sarath Publications

A current view of C₄ and CAM evolution holds that all of the constituent enzymes of the C₄ pathway preexisted in C₃ plants, albeit functioning in nonphotosynthetic capacities.

Effects Of Antimetabolites On Sclerotium Rolfsii Growth And Oxalic Acid Production, Ezikiel Okafor-Agilo

Seton Hall University Dissertations and Theses (ETDs)

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Immobolisation And Biochemical Characterisation Of A Selected Commercially Available Laccase, Chandra Sundaralingam

Student Works (2000-2009)

ln this study, laccase from Novo Nordisk was chosen for biochemical characterization and immobilization investigation. The specific aims of this work was to develop optimum conditions for immobilization of laccase by entrapment method using copper alginate and characterize the laccase biochemically. The pH optimum of the crude enzyme extract was 5.5 and was found to be stable over a wide range of pH values. The apparent molecular weight of this enzyme was around 79,000 Daltons. Three peaks of activity (isoenzymes) were obtained from isoelectric focusing of pH values 4.7, 5.2 and 6.0. The optimum pH of the immobilized laccase by …

Identification Of The Domains Of Urer, An Arac-Like Transcriptional Regulator Of The Urease Gene Cluster In Proteus Mirabilis, Carrie A. Poore, Christopher Coker, Jonathan D. Dattelbaum, Harry L.T. Mobley

Chemistry Faculty Publications

Proteus mirabilis urease catalyzes the hydrolysis of urea to CO₂ and NH₃, resulting in urinary stone formation in individuals with complicated urinary tract infections. UreR, a member of the AraC family, activates transcription of the genes encoding urease enzyme subunits and accessory proteins, ureDABCEFG, as well as its own transcription in the presence of urea. Based on sequence homology with AraC, we hypothesized that UreR contains both a dimerization domain and a DNA-binding domain. A translational fusion of the leucine zipper dimerization domain (amino acids 302 to 350) of C/EBP and the C-terminal half of UreR …

Group Viii Spiroplasma Of Costa Rica, Kimberly M. Stewart

College of Graduate Studies: Theses & Dissertations (1964–2006)

Spiroplasma bacteria isolates are classified or identified through a series of serological tests that normally consists of screening, one-way deformation tests, cloning, antisera production, and reciprocal deformation tests. Serological tests on the spiroplasmas are followed by molecular analysis. The standard molecular analysis used for spiroplasmas has been 16S rRNA sequencing. The primary goal ofmy research was to evaluate serologically Group VIII spiroplasmas isolated from tropical Costa Rican tabanids (Diptera: Tabanidae) and to compare them to the temperate North American Group VIII spiroplasmas. A secondary goal was to evaluate both the temperate and tropical Group VIII strains by sequencing the 16S-23S …

An Investigation Of Captive Florida Manatee (Trichecus Manatus Latirostris) Behavior And Social Interactions, Jennifer Sadler Young

College of Graduate Studies: Theses & Dissertations (1964–2006)

Human impacts such as pollution, habitat alteration, and boating have caused many injuries and deaths of the Florida manatee, Trichechus manutus latirostris. Rehabilitation centers have been established in Florida to help return sick or injured manatees back to the wild. Manatees that are released back to the wild after undergoing rehabilitation in captivity might lose some of their abilities to survive in the wild. A factor that might alter behavior in captive individuals was examined by determining if length of time spent in captivity influenced the behavior of manatees during rehabilitation. Even though all facilities follow similar guidelines, invariable …

The Prevalence Of Human Granulocytic Ehrlichiae In Ixodes Scapularis Ticks In Southeastern Georgia, Heather Anne Merten

College of Graduate Studies: Theses & Dissertations (1964–2006)

Human granulocytic ehrlichiosis (HGE) is a newly emerging, tick-borne zoonosis in North America. The agent that causes HGE is very similar to Ehrlichia phagocytophilia and Ehrlichia equi, but has yet to be classified as a separate species.

This study was carried out in order to determine the prevalence rate of HGE infection in blacklegged ticks (Ixodes scapularis) in insular and mainland sites in southeastern Georgia using nested PCR techniques. Four locations were studied representing 2 barrier islands and 2 mainland sites. We hypothesized that areas that have low levels of human disturbance and are somewhat isolated will …

A Comparative Study Of The Reproductive Biology Of Sympatric Morning Glories (Ipomoea-Convolvulaceae), Dexter R. Sowell Ii

College of Graduate Studies: Theses & Dissertations (1964–2006)

Despite the astounding diversity of flowering plants and of mobile organisms that serve as pollinators, sympatric co-flowering plants can potentially share pollinators with each other. This places two selection pressures on plants. First, plants compete for pollinators to visit their flowers and secondly, entice pollinators to be faithful (specialized) and visit only other members of their species. I tested whether sympatric co-flowering plants reduced their selection pressures via temporal partitioning or differential attraction of pollinators using six closely related, sympatric co-flowering morning glories (Ipomoea-Convolvulaceae).

Diurnal flower phenology tightly overlapped, which did not facilitate temporal partitioning of pollinators. However, …

Control Of Phytoplankton Growth By Iron And Silicic Acid Availability In The Subantarctic Ocean: Experimental Results From The Saz Project, D. A. Hutchins, Peter N. Sedwick, G. R. Ditullio, P. W. Boyd, B. Queguiner, F. B. Griffiths, C. Crossley

OES Faculty Publications

Subantarctic Southern Ocean surface waters in the austral summer and autumn are characterized by high concentrations of nitrate and phosphate but low concentrations of dissolved iron (Fe, similar to0.05 nM) and silicic acid (Si, <1 muM). During the Subantarctic Zone AU9706 cruise in March 1998 we investigated the relative importance of Fe and Si in controlling phytoplankton growth and species composition at a station within the subantarctic water mass (46.8degreesS, 142degreesE) using shipboard bottle incubation experiments. Treatments included unamended controls; 1.9 nM added iron (+Fe); 9 muM added silicic acid (+Si); and 1.9 nM addediron plus 9 muM added silicic acid (+Fe+Si). We followed a detailed set of biological and biogeochemical parameters over 8 days. Fe added alone clearly increased community growth rates and nitrate drawdown and altered algal community composition relative to control treatments. Surprisingly, small, lightly silicified pennate diatoms grew when Fe was added either with or without Si, despite the extremely low ambient silicic acid concentrations. Pigment analyses suggest that lightly silicified chrysophytes (type 4 haptophytes) may have preferentially responded to Si added either with or without Fe. However, for many of the parameters measured the +Fe+Si treatments showed large increases relative to both the +Fe and +Si treatments. Our results suggest that iron is the proximate limiting nutrient for chlorophyll production, photosynthetic efficiency, nitrate drawdown, and diatom growth, but that Si also exerts considerable control over algal growth and species composition. Both nutrients together are needed to elicit a maximum growth response, suggesting that both Fe and Si play important roles in structuring the subantarctic phytoplankton community.

Effects Of Iron, Silicate, And Light On Dimethylsulfoniopropionate Production In The Australian Subantarctic Zone, G. R. Ditullio, P. N. Sedwick, D. R. Jones, P. W. Boyd, A. C. Crossley, D. A. Hutchins

OES Faculty Publications

Shipboard bottle incubation experiments were performed to investigate the effects of iron, light, and silicate on algal production of particulate dimethylsulfoniopropionate (DMSP_p) in the Subantarctic Zone (SAZ) south of Tasmania during March 1998. Iron enrichment resulted in threefold to ninefold increases in DMSP_p concentrations relative to control treatments, following 7 and 8-day incubation experiments. Additions of Fe and Si preferentially stimulated the growth of lightly-silicified pennate diatoms and siliceous haptophytes, respectively, to which we attribute the increased DMSP_p production in the incubation bottles. Both of these algal groups were previously believed to be low DMSP_{p …}

Characterization Of The Human Cytomegalovirus Ul75 Late Gene Promoter, Bernard J. P. Mcwatters

Theses and Dissertations in Biomedical Sciences

Gene expression during productive infection by the human cytomegalovirus (HCMV) occurs in an ordered and sequential manner, beginning with immediate early (IE), then early (E) and finally late (L) gene expression. Significant work has addressed the regulation of IE and E gene expression while relatively little work has addressed the control of late gene expression. In order to further address HCMV late gene expression, the promoter of the HCMV UL75 (glycoprotein H, gH) late gene was characterized. The data obtained in this study were combined with observations made in two other studies that have addressed HCMV late gene expression to …

Characterization Of The Biological Activities Of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (Gfp/Hzp3), Zhiyong Lin

Theses and Dissertations in Biomedical Sciences

Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA).

This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell …