Biochemistry, Biophysics, and Structural Biology Commons^™

Challenges And Opportunities For Toxicology In Mexico, Rodrigo Franco, Balam Muñoz

School of Veterinary and Biomedical Sciences: Faculty Publications

We have developed this Special Issue titled “Challenges and Opportunities for Toxicology in Mexico” with the aim to highlight not only the challenges toxicology research faces in Mexico, but also the efforts and drive towards cutting edge research to address them. Altogether, this special issue should be a valuable source of information for all readers with an interest in toxicology and human health.

Mexico is an important economy worldwide and is a major exporter of several supplies and commodities. Because of this, its industry is a source of occupational exposure to several chemical derivatives (Nieusma 2011). The situation of occupational …

Computer Simulation Of A Hollow-Fiber Bioreactor: Heparan Regulated Growth Factors-Receptors Binding And Dissociation Analysis, Changjiang Zhang

University of Kentucky Doctoral Dissertations

This thesis demonstrates the use of numerical simulation in predicting the behavior of proteins in a flow environment.

A novel convection-diffusion-reaction computational model is first introduced to simulate fibroblast growth factor (FGF-2) binding to its receptor (FGFR) on cell surfaces and regulated by heparan sulfate proteoglycan (HSPG) under flow in a bioreactor. The model includes three parts: (1) the flow of medium using incompressible Navier-Stokes equations; (2) the mass transport of FGF-2 using convection-diffusion equations; and (3) the cell surface binding using chemical kinetics. The model consists of a set of coupled nonlinear partial differential equations (PDEs) for flow and …

Fabrication And Characterization Of Mesoscale Protein Patterns Using Atomic Force Microscopy (Afm), Pei Gao

University of Kentucky Doctoral Dissertations

A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing.

As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It …

Characterization Of G10h Promoter And Isolation Of Wrky Transcription Factors Involved In Catharanthus Terpenoid Indole Alkaloid Biosynthesis Pathway, Nitima Suttpanta

University of Kentucky Doctoral Dissertations

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semi-synthetic anticancer drugs. Biosynthesis of TIAs is tissue-specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. This study reports the isolation and characterization of the G10H promoter and two WRKY transcription factors regulating TIA biosynthesis.

Geraniol 10-hydroxylase (G10H) controls the first committed step in the biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a …

Crystallization And Preliminary X-Ray Crystallographic Analysis Of Ligand-Free And Arginine-Bound Forms Of Thermotoga Maritima Arginine-Binding Protein, Alessia Ruggiero, Jonathan D. Dattelbaum, Anna Pennacchio, Luisa Iozzion, Maria Staiano, Matthew S. Luchansky, Bryan S. Der, Rita Berisio, Sabato D'Auria, Luigi Vitagliano

Chemistry Faculty Publications

The arginine-binding protein from Thermotoga maritima (TmArgBP) is an arginine-binding component of the ATP-binding cassette (ABC) transport system in this hyperthermophilic bacterium. This protein is endowed with an extraordinary stability towards thermal and chemical denaturation. Its structural characterization may provide useful insights for the clarification of structure– stability relationships and for the design of new biosensors. Crystallization trials were set up for both arginine-bound and ligand-free forms of TmArgBP and crystals suitable for crystallographic investigations were obtained for both forms. Ordered crystals of the arginine adduct of TmArgBP could only be obtained by using the detergent LDAO as an additive …

Biologically Active Alkaloids From Kopsia., Yap Wai Sum

Student Works (2010-2019)

Two Malaysian plants viz., Kopsia pauciflora and Kopsia grandifolia were investigated for their alkaloidal constituents. A total of 40 alkaloids were isolated and characterized from K. pauciflora, of which 12 are new alkaloids. The new alkaloids isolated include seven aspidofractinine alkaloids (1, 2, 3, 4, 5, 6, 7), and five eburnane alkaloids (8, 9, 10, 11, 12). K. grandifolia yielded a total of eight alkaloids. Of these, three are new. The new alkaloids are grandilodine A (41), grandilodine B (42), and grandilodine C (43). Grandilodine A (41), grandilodine C (43), and lapidilectine B (47) were found to reverse multidrug resistance …

Quantifying Agonist Activity At G Protein-Coupled Receptors, Frederick J. Ehlert, Hinako Suga, Michael T. Griffin

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.

Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence …

Preparation And Crystal Structures Of Chiral And Non-Chiral Mixed Ligand Copper Complexes Containing N-Methyl Imidazole And Various N-Phthaloylalanines, Hiral Patel

Theses, Dissertations and Culminating Projects

A quasiracemate is the cocrystallization of two similarly shaped but chemically distinct molecules of opposite handedness. There has been extensive work done in the field of organic quasiracemates by Dr. Kraig A. Wheeler. Limited work has been done in inorganic quasiracemates. The goal of this study was to prepare various metal based enantiomers, racemates, and quasiracemates using A-phthaloylalanines, and TV-methylimidazole as ligands bound to copper arranged in a centrosymmetric pattern. In this study, we developed a method to prepare A-phthaloylalanines and its copper complexes. Three complexes have yielded crystal structures: a racemate, bis(7V-methylimidazole) bis(D,L-3-chloro-7V-phthaloylalanato)copper(II); a quasiracemate, bis(A-methyhmidazole)(L-3-chloro-iV-phthaloylalanato)(D-3-methyl-7V-phthaloylalanato) copper(II); and an …

Rate-Limiting Step Of Cone Phototransduction Recovery And Oguchi Disease Mechanisms, Frank Chen

Theses and Dissertations

ABSTRACT RATE-LIMITING STEP OF CONE PHOTOTRANSDUCTION RECOVERY AND OGUCHI DISEASE MECHANISMS By Frank Sungping Chen Advisor: Ching-Kang Jason Chen, Ph.D. Retinal photoreceptors provide the first gateway in which light information from the environment is transformed into neuronal signals. The cone and rod photoreceptors are responsible for day and night vision, respectively. Understanding rod and cone phototransduction is to figure out how these cells differ in their temporal and spatial sensitivities to allow perception of a broad dynamic range of stimuli. Phototransduction is mediated through a Gprotein signaling cascade. Light absorption by visual pigment triggers the isomerization of 11- cis-retinal covalently …

A Messenger Molecule Governs Interdependency In An Evolved Pseudomonas Aeruginosa Biofilm Community, Thomas Macelliott Johnson

Honors Theses and Capstones

Biofilm populations are known to harbor great diversity, but the importance of this diversity is not fully understood. A likely contributor to this variation is the second messenger molecule cyclic-di-GMP: low levels associate with a planktonic lifestyle while high levels favor biofilm formation. In ongoing studies of an evolving biofilm population of Pseudomonas aeruginosa (PA), we observed extensive ecological diversification that may relate to this lifestyle switch. In PA, the gene bifA encodes a phosphodiesterase that is known to degrade cyclic-di-GMP and reduce biofilm. This gene was cloned onto a plasmid under control of an inducible promoter and the plasmid …

Identification And Characterization Of Fkbp52-Specific Inhibitors For The Treatment Of Prostate Cancer, Johanny Tonos De Leon

Open Access Theses & Dissertations

Steroid hormone receptors require the ordered assembly of various chaperone and cochaperone proteins in order to reach a functional state. The final stage in the receptor maturation process requires the formation of a mutimeric complex consisting of Hsp90 dimer, p23, and one of several large immunophilins. Studies conducted previously demonstrated that the large immunophilin FKBP52 acts to potentiate glucocorticoid, androgen, and progesterone receptor signaling pathways. The aim of these studies was to identify and characterize FKBP52-specific inhibitors that would not only serve as tools for the pharmacological analysis of FKBP52-receptor interactions, but may also lead to novel drugs with significant …

The Role Of Mdm2 In Estrogen-Mediated Breast Cancer Cell Proliferation, Angelika Brekman

Dissertations, Theses, and Capstone Projects

Estrogen signaling is important in breast cancer development and progression. Mdm2, a negative regulator of the p53 tumor suppressor, is often over-expressed in estrogen receptor positive breast cancers. To study the role of Mdm2 in the estrogen-mediated breast cancer cell proliferation, we examined the effect of estrogen on the p53-Mdm2 pathway in estrogen receptor positive and p53 wild-type MCF-7 breast cancer cells. Estrogen-mediated increase in cell proliferation correlated with increased Mdm2, but no concomitant decrease in the p53 protein level. Blocking Mdm2 expression with inducible shRNA inhibited estrogen-mediated cell proliferation and colony formation in soft agar. Mdm2 knockdown in the …

An Upper Limit For Macromolecular Crowding Effects, Andrew C. Miklos, Congang Li, Courtney D. Sorell, L. Andrew Lyon, Gary J. Pielak

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Background: Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is similar to 2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of similar to 300 nm.

Results: Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild …

Investigation Of Antioxidant And Cytotoxic Activities Of Portulaca Oleracea., Ong Kiah Ju

Student Works (2010-2019)

Portulaca oleracea known as purslane in English or Gelang Pasir in Bahasa Melayu. Traditionally, this plant is used to alleviate pain and swelling. This plant is reported to exhibit many pharmacological effects such as antibacterial, analgesic, antiinflammatory and wound healing. In the present study, the experimental approach was based on bioassay-guided fractionation. The crude methanolic and fractionated extracts were initially investigated for their antioxidant and cytotoxic activities. The cytotoxic effects of the extracts were tested against five human cell lines namely the hormonedependent breast carcinoma cell line (MCF7), human nasopharyngeal cell line (KB), human cervical carcinoma cell line (Ca Ski), …

Hydrolysis Of Amelogenin By Matrix Metalloprotease-20 Accelerates Mineralization In Vitro, Vuk Uskoković, Feroz Khan, Haichuan Liu, Halina Ewa Witkowska, Li Zhu, Wu Li, Stefan Habelitz

Pharmacy Faculty Articles and Research

In the following respects, tooth enamel is a unique tissue in the mammalian body: (a) it is the most mineralized and hardest tissue in it comprising up to 95 wt% of apatite; (b) its microstructure is dominated by parallel rods composed of bundles of 40 – 60 nm wide apatite crystals with aspect ratios reaching up to 1:10,000 and (c) not only does the protein matrix that gives rise to enamel guides the crystal growth, but it also conducts its own degradation and removal in parallel. Hence, when mimicking the process of amelogenesis in vitro, crystal growth has to …

Biomimetic Precipitation Of Uniaxially Grown Calcium Phosphate Crystals From Full-Length Human Amelogenin Sols, Vuk Uskoković, Wu Li, Stefan Habelitz

Pharmacy Faculty Articles and Research

Human dental enamel forms over a period of 2 – 4 years by substituting the enamel matrix, a protein gel mostly composed of a single protein, amelogenin with fibrous apatite nanocrystals. Self-assembly of a dense amelogenin matrix is presumed to direct the growth of apatite fibers and their organization into bundles that eventually comprise the mature enamel, the hardest tissue in the mammalian body. This work aims to establish the physicochemical and biochemical conditions for the synthesis of fibrous apatite crystals under the control of a recombinant fulllength human amelogenin matrix in combination with a programmable titration system. The growth …

Dynamic Light Scattering And Zeta Potential Of Colloidal Mixtures Of Amelogenin And Hydroxyapatite In Calcium And Phosphate Rich Ionic Milieus, Vuk Uskoković, Roselyn Odsinada, Sonia Djordjevic, Stefan Habelitz

Pharmacy Faculty Articles and Research

The concept of zeta-potential has been used for more than a century as a basic parameter in controlling the stability of colloidal suspensions, irrespective of the nature of their particulate ingredients – organic or inorganic. There are prospects that self-assembly of peptide species and the protein-mineral interactions related to biomineralization may be controlled using this fundamental physicochemical parameter. In this study, we have analyzed the particle size and zeta-potential of the full-length recombinant human amelogenin (rH174), the main protein of the developing enamel matrix, in the presence of calcium and phosphate ions and hydroxyapatite (HAP) particles. As calcium and phosphate …

Altered Calcium Handling In Cerebellar Purkinje Neurons With The Malignant Hyperthermia Mutation, Ryr1-Y522s/+, George C. Talbott

Electronic Theses and Dissertations

To investigate the etiology of malignant hyperthermia and central core disease, mouse models have recently been generated and characterized (Chelu et al., 2006). These RyRY522S/+ knock-in mutant mice provide an excellent tool to investigate calcium dysregulation, its pathological consequences, and potential therapeutic approaches. Skeletal muscle harboring this mutation exhibits calcium leak from internal stores and an increased sensitivity to activation by caffeine, voltage, and temperature (Durham et al., 2008). Although alterations in RyR1 channel function and resultant changes in cellular function have been characterized in skeletal muscle, the effects of MH mutations in RyR1 on central nervous system function have …

Determination Of The Effects Of Ficus Deltoidea Fractions With Antioxidative And Anticancer Properties On The Proteome Profile Of Selected Cancer Cell Lines., Yew Han Choi

Student Works (2010-2019)

Ficus deltoidea’s extracts (three types of leaves and two types of fruits) were prepared through boiling method followed by semi-purification with ammonium sulfate precipitation at 30, 60 and 90% saturation. The samples were evaluated for their antioxidative activities and cytotoxicity towards three cell lines (Hep G2, Ca Ski and Chang Liver cells). Antioxidants results indicated that large type leaf (LL) and medium type leaf (ML) extracts were better compared to small type leaf (SL) while big type fruit (BF) was better compared to small type fruit (SF). Meanwhile, only SF semi-purified fractions showed cytotoxic effects towards the cell lines. Therefore, …