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Altered Self-Assembly And Apatite Binding Of Amelogenin Induced By N-Terminal Proline Mutation, Li Zhu, Vuk Uskoković, Thuan Le, Pamela DenBesten, Yulei Huang, Stefan Habelitz, Wu Li 2011 University of California - San Francisco

Altered Self-Assembly And Apatite Binding Of Amelogenin Induced By N-Terminal Proline Mutation, Li Zhu, Vuk Uskoković, Thuan Le, Pamela Denbesten, Yulei Huang, Stefan Habelitz, Wu Li

Pharmacy Faculty Articles and Research

Objective—A single Pro-70 to Thr (p.P70T) mutation of amelogenin is known to result in hypomineralized amelogenesis imperfecta (AI). This study aims to test the hypothesis that the given mutation affects the self-assembly of amelogenin molecules and impairs their ability to conduct the growth of apatite crystals.

Design—Recombinant human full-length wild-type (rh174) and p.P70T mutated amelogenins were analyzed using dynamic light scattering (DLS), protein quantification assay and atomic force microscopy (AFM) before and after the binding of amelogenins to hydroxyapatite crystals. The crystal growth modulated by both amelogenins in a dynamic titration system was observed using AFM ...


Mechanisms Of Decreased Cholesterol Absorption Mediated By Phytosterols In The Intestinal Lumen, Andrew W. Brown 2011 University of Nebraska-Lincoln

Mechanisms Of Decreased Cholesterol Absorption Mediated By Phytosterols In The Intestinal Lumen, Andrew W. Brown

Nutrition & Health Sciences Dissertations & Theses

Phytosterols and their fatty acyl esters have been known for decades to lower LDL cholesterol, making them powerful nutraceuticals in lowering cardiovascular disease risk. The mechanisms by which phytosterols lower cholesterol, though, have been incompletely characterized. Three studies were executed to examine three aspects of cholesterol and phytosterol interactions in the intestinal lumen. In the first study, the ability of pancreatic cholesterol esterase to hydrolyze phytosterol esters was examined. Pancreatic cholesterol esterase hydrolyzed phytosterol esters, but the rate of hydrolysis proved sensitive to the structures of both the sterol and ester components. In the second study, cholesterol micellarization was challenged ...


Role Of Protein Kinase C-Iota In Glioblastoma, Shraddha R. Desai 2011 University of South Florida

Role Of Protein Kinase C-Iota In Glioblastoma, Shraddha R. Desai

Graduate Theses and Dissertations

The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma.

The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of ...


Targeted Deletion Of The Mouse Mitoferrin1 Gene: From Anemia To Protoporphyria, Marie-Berengere Troadec, David Warner, Jared Wallace, Kirk Thomas, Gerald J. Spangrude, John Phillips, Oleh Khalimonchuk, Barry H. Paw, Diane McVey Ward, Jerry Kaplan 2011 University of Utah

Targeted Deletion Of The Mouse Mitoferrin1 Gene: From Anemia To Protoporphyria, Marie-Berengere Troadec, David Warner, Jared Wallace, Kirk Thomas, Gerald J. Spangrude, John Phillips, Oleh Khalimonchuk, Barry H. Paw, Diane Mcvey Ward, Jerry Kaplan

Biochemistry -- Faculty Publications

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show ...


A New Noncovalent Force: Comparison Of P∙∙∙N Interaction With Hydrogen And Halogen Bonds, Steve Scheiner 2011 Utah State University

A New Noncovalent Force: Comparison Of P∙∙∙N Interaction With Hydrogen And Halogen Bonds, Steve Scheiner

Chemistry and Biochemistry Faculty Publications

When PH(3) is paired with NH(3), the two molecules are oriented such that the P and N atoms face one another directly, without the intermediacy of a H atom. Quantum calculations indicate that this attraction is due in part to the transfer of electron density from the lone pair of the N atom to the σ(∗) antibond of a P-H covalent bond. Unlike a H-bond, the pertinent hydrogen is oriented about 180° away from, instead of toward, the N, and the N lone pair overlaps with the lobe of the P-H σ(∗) orbital that is closest to the ...


An Apoptosis Targeted Stimulus With Nanosecond Pulsed Electric Fields (Nspefs) In E4 Squamous Cell Carcinoma, Wei Ren, Stephen J. Beebe 2011 Old Dominion University

An Apoptosis Targeted Stimulus With Nanosecond Pulsed Electric Fields (Nspefs) In E4 Squamous Cell Carcinoma, Wei Ren, Stephen J. Beebe

Bioelectrics Publications

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was [95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels ...


Hydrogen Peroxide Probes Directed To Different Cellular Compartments, Mikalai Malinouski, You Zhou, Vsevolod V. Belousov, Dolph L. Hatfield, Vadim N. Gladyshev 2011 Harvard Medical School

Hydrogen Peroxide Probes Directed To Different Cellular Compartments, Mikalai Malinouski, You Zhou, Vsevolod V. Belousov, Dolph L. Hatfield, Vadim N. Gladyshev

Biochemistry -- Faculty Publications

Background: Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells.

Principal Findings: Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed ...


Mne1 Is A Novel Component Of The Mitochondrial Splicing Apparatus Responsible For Processing Of A Cox1 Group I Intron In Yeast, Talina Watts, Oleh Khalimonchuk, Rachel Z. Wolf, Edward M. Turk, Georg Mohr, Dennis R. Winge 2011 University of Utah Health Sciences Center

Mne1 Is A Novel Component Of The Mitochondrial Splicing Apparatus Responsible For Processing Of A Cox1 Group I Intron In Yeast, Talina Watts, Oleh Khalimonchuk, Rachel Z. Wolf, Edward M. Turk, Georg Mohr, Dennis R. Winge

Biochemistry -- Faculty Publications

Saccharomyces cerevisiae cells lacking Mne1 are deficient in

intron splicing in the gene encoding the Cox1 subunit of cytochrome

oxidase but contain wild-type levels of the bc1 complex.

Thus, Mne1 has no role in splicing of COB introns or expression

of the COB gene. Northern experiments suggest that splicing of

the COX1 aI5β intron is dependent on Mne1 in addition to the

previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing

of the aI5_ intron is similarly impaired in mne1∆ and

mrs1∆ cells and overexpression of Mrs1 partially restores the

respiratory function of mne1∆ cells. Mrs1 is known to ...


Methods Of Protein Characterization, Michael Tieu 2011 University of Puget Sound

Methods Of Protein Characterization, Michael Tieu

Summer Research

AppA is a protein in Rhodobacter sphaeroides that has been the topic of debate among scientists for the past several years with regards to the structure of the protein. It has been known that AppA has an effect on the activity of PpsR, which controls the gene expression of photosystems. There are two conflicting experimental structures (2IYG and 1YRX) of the protein, both of which claim to be taken in the dark phase (meaning when there is no light shining on the protein). The debate is about whether some slight differences in the structures represent the shift from the dark ...


Exploration Of The Active Site Specificity Of Mala, A Glucosidase From The Predatory Bacterium Bdellovibrio Bacteriovorus, Christine Isabella 2011 University of Puget Sound

Exploration Of The Active Site Specificity Of Mala, A Glucosidase From The Predatory Bacterium Bdellovibrio Bacteriovorus, Christine Isabella

Summer Research

Sequencing of the HD100 genome of Bdellovibrio Bacteriovorus in 2005 revealed a gene for the putative maltase, MalA. However, given the bacterium’s observed disuse of prey carbohydrates as an energy source, this enzyme is seemingly out of place. In this study, the specificity and activity of MalA were explored through various enzymatic assays. Using p-nitrophenol-α-D-glucopyranoside (p-NPG) as a colorimetric substrate to allow for rapid and accurate detection of enzymatic activity through spectrophotometry, enzyme stability inhibition of p-NPG cleavage were explored. While numerous alpha-linked disaccharides were shown to inhibit MalA, only maltose was shown to be cleaved into glucose.


Investigating The Bacterial Predator Bdellovibrio’S Ability To Degrade Aspartate, Scott Anderson 2011 University of Puget Sound

Investigating The Bacterial Predator Bdellovibrio’S Ability To Degrade Aspartate, Scott Anderson

Summer Research

Bdellovibrio bacteriovorus is a predatory Gram-negative Deltaproteobacterium that attacks and invades larger Gram-negative bacteria devouring them from within (Sockett, 2004). Enzymatic results obtained in the 1970s suggest that Bdellovibrio relies on its tricarboxylic acid (TCA) cycle and the oxidation of prey cell derived amino acids (Hespell, 1976). However, annotation of the published genome of Bdellovibrio HD100 revealed that it lacked numerous genes involved with the degradation of amino acids (Rendulic, 2004). Thus it is of great interest to determine if Bdellovibrio can degrade amino acids. If it can, new genes related to the degradation of amino acids will be discovered ...


Developing A Protocol For Purifying The Mala Enzyme In Bdellovibrio Bacteriovorus, John Jared Trecker 2011 University of Puget Sound

Developing A Protocol For Purifying The Mala Enzyme In Bdellovibrio Bacteriovorus, John Jared Trecker

Summer Research

The sequenced genome of the gram-negative predatory bacterium Bdellovibrio bacteriovorus contains a gene that encodes for malA, a putative maltase. Given the bacterium's observed disuse of prey carbohydrates, the gene's presence is mysterious. That characterization of the enzyme and studies of its activity and specificity can be better carried out, it is necessary to obtain pure enzyme. Protein was collected from lysed cultures of Top10/pmalA E. coli. Attempted purification by ion-exchange chromatography with DEAE columns produced significantly purer protein; SP ion exchange columns were unsuccessful, as were heparin and hydroxyapatite affinity columns. Gel filtration chromatography should prove ...


Lysophosphatidic Acid Production And Signaling In Platelets, Zachary Bennett Fulkerson 2011 University of Kentucky

Lysophosphatidic Acid Production And Signaling In Platelets, Zachary Bennett Fulkerson

Theses and Dissertations--Physiology

Lysophosphatidic acid (LPA) belongs to a class of extracellular lipid signaling molecules. In the vasculature, LPA may regulate platelet activation and modulate endothelial and smooth muscle cell function. LPA has therefore been proposed as a mediator of cardiovascular disease.

The bulk of circulating LPA is produced from plasma lysophosphatidylcholine (LPC) by autotaxin (ATX), a secreted lysophospholipase D (lysoPLD). Early studies suggest that some of the production of circulating LPA is platelet-dependent. ATX possesses an N-terminal somatomedin B-like domain suggesting the hypothesis that ATX interacts with platelet integrins which may localize ATX to substrate in the membrane and/or alter the ...


Role Of Non-Coding Rna Nc4 In Mll And Cyp33 Mediated Regulation Of Hoxc8, Jessica Arvindbhai Solanki 2011 Loyola University Chicago

Role Of Non-Coding Rna Nc4 In Mll And Cyp33 Mediated Regulation Of Hoxc8, Jessica Arvindbhai Solanki

Dissertations

MLL or Mixed Lineage Leukemia gene is clinically known for its involvement in genetic translocation with more than 70 different partners identified each giving rise to a highly leukemogenic fusion protein. With a poor prognosis of MLL related leukemia, an investigation into its role during hematopoiesis has been a very active field of research. In order to design strategies to combat the MLL leukemia, it becomes essential to delineate the molecular mechanisms behind the function of MLL wild type protein. Wild type MLL is a transcriptional maintenance protein from the Trithorax Group (TrxG) that resides in complex with other chromatin ...


Lead Identification Of Β-Lactam And Related Imine Inhibitors Of The Molecular Caperone Heat Shock Protein 90, Niamh O'Boyle, Andrew JS Knox, Trevor P. Price, D. Clive Williams, Daniela M. Zisterer, David G. Lloyd, Mary J. Meegan 2011 Technological University Dublin

Lead Identification Of Β-Lactam And Related Imine Inhibitors Of The Molecular Caperone Heat Shock Protein 90, Niamh O'Boyle, Andrew Js Knox, Trevor P. Price, D. Clive Williams, Daniela M. Zisterer, David G. Lloyd, Mary J. Meegan

Articles

Heat shock protein 90 is an emerging target for oncology therapeutics. Inhibitors of this molecular chaperone, which is responsible for the maintenance of a number of oncogenic proteins, have shown promise in clinical trials and represent a new and exciting area in the treatment of cancer. Heat shock protein 90 inhibitors have huge structural diversity, and here we present the identification of inhibitors based on β-lactam and imine templates. β-Lactam 5 and imines 12 and 18 exhibit binding to heat shock protein 90-α with IC50 values of 5.6 μM, 14.5 μM and 22.1 μM respectively. The ...


Synthesis, Biochemical And Molecular Modelling Studies Of Antiproliferative Azetidinones Causing Microtubule Disruption And Mitotic Catastrophe, Niamh O'Boyle, Miriam Carr, Lisa M. Greene, Niall O. Keely, Andrew JS Knox, Thomas McCabe, David G. Lloyd, Daniela M. Zisterer, Mary J. Meegan 2011 Technological University Dublin

Synthesis, Biochemical And Molecular Modelling Studies Of Antiproliferative Azetidinones Causing Microtubule Disruption And Mitotic Catastrophe, Niamh O'Boyle, Miriam Carr, Lisa M. Greene, Niall O. Keely, Andrew Js Knox, Thomas Mccabe, David G. Lloyd, Daniela M. Zisterer, Mary J. Meegan

Articles

The structure-activity relationships of antiproliferative β-lactams, focusing on modifications at the 4-position of the β-lactam ring, is described. Synthesis of this series of compounds was achieved utilizing the Staudinger and Reformatsky reactions. The antiproliferative activity was assessed in MCF-7 cells, where the 4-(4-ethoxy)phenyl substituted compound 26 displayed the most potent activity with an IC50 value of 0.22 μM. The mechanism of action was demonstrated to be by inhibition of tubulin. Cell exposure to combretastatin A-4 and 26 led to arrest of MCF-7 cells in the G2/M phase of the cell cycle and induction of apoptosis ...


Targeted Identification Of Metastasis-Associated Cell-Surface Sialoglycoproteins In Prostate Cancer, Lifang Yang, Julius O. Nyalwidhe, Sigi Guo, Richard R. Drake, O. John Semmes 2011 Old Dominion University

Targeted Identification Of Metastasis-Associated Cell-Surface Sialoglycoproteins In Prostate Cancer, Lifang Yang, Julius O. Nyalwidhe, Sigi Guo, Richard R. Drake, O. John Semmes

Bioelectrics Publications

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC4ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins ...


Electrostatic Effects On (Di)Terpene Synthase Product Outcome, Ke Zhou, Reuben J. Peters 2011 Iowa State University

Electrostatic Effects On (Di)Terpene Synthase Product Outcome, Ke Zhou, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Terpene synthases catalyze complex reactions, often forming multiple chiral centers in cyclized olefin products from acyclic allylic diphosphate precursors, yet have been suggested to exert little control over the actual reaction, instead largely serving as inert templates. However, recent results highlight stereoelectronic effects exerted by these enzymes. Perhaps not surprisingly, the pyrophosphate co-product released in the initiating and rate-limiting chemical step provides an obvious counter-ion that may drive carbocation migration towards itself. This is emphasized by the striking effects of a recently uncovered single residue switch for diterpene synthase product outcome, whereby substitution of hydroxyl residues for particular aliphatic residues ...


Structure And Mechanism Of The Diterpene Cyclase Ent-Copalyl Diphosphate Synthase, Mustafa Köksal, Huayou Hu, Robert M. Coates, Reuben J. Peters, David D. Christianson 2011 University of Pennsylvania

Structure And Mechanism Of The Diterpene Cyclase Ent-Copalyl Diphosphate Synthase, Mustafa Köksal, Huayou Hu, Robert M. Coates, Reuben J. Peters, David D. Christianson

Biochemistry, Biophysics and Molecular Biology Publications

The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.


Immunoglobulin Structure Exhibits Control Over Cdr Motion, Michael T. Zimmermann, Aris Skliros, Andrzej Kloczkowski, Robert L. Jernigan 2011 Iowa State University

Immunoglobulin Structure Exhibits Control Over Cdr Motion, Michael T. Zimmermann, Aris Skliros, Andrzej Kloczkowski, Robert L. Jernigan

Biochemistry, Biophysics and Molecular Biology Publications

Motions of the IgG structure are evaluated using normal mode analysis of an elastic network model to detect hinges, the dominance of low frequency modes, and the most important internal motions. One question we seek to answer is whether or not IgG hinge motions facilitate antigen binding. We also evaluate the protein crystal and packing effects on the experimental temperature factors and disorder predictions. We find that the effects of the protein environment on the crystallographic temperature factors may be misleading for evaluating specific functional motions of IgG. The extent of motion of the antigen binding domains is computed to ...


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