Activation of T cells by carbamazepine and carbamazepine metabolites

BACKGROUND: T-cell-mediated hypersensitivity is a rare but serious manifestation of drug therapy. OBJECTIVES: To explore the mechanisms of drug presentation to T cells and the possibility that generation of metabolite-specific T cells may provoke cross-sensitization between drugs. METHODS: A lymphocyte transformation test was performed on 13 hypersensitive patients with carbamazepine, oxcarbazepine, and carbamazepine metabolites. Serial dilution experiments were performed to generate drug (metabolite)-specific T-cell clones to explore the structural basis of the T-cell response and mechanisms of antigen presentation. 3-Dimensional energy-minimized structures were generated by using computer modeling. The role of drug metabolism was analyzed with 1-aminobenzotriazole. RESULTS: Lymphocytes and T-cell clones proliferated with carbamazepine, oxcarbazepine, and some (carbamazepine 10,11 epoxide, 10-hydroxy carbamazepine) but not all stable carbamazepine metabolites. Structure activity studies using 29 carbamazepine (metabolite)-specific T-cell clones revealed 4 patterns of drug recognition, which could be explained by generation of preferred 3-dimensional structural conformations. T cells were stimulated by carbamazepine (metabolites) bound directly to MHC in the absence of processing. The activation threshold for T-cell proliferation varied between 5 minutes and 4 hours. 1-Aminobenzotriazole, which inhibits cytochrome P450 activity, did not prevent carbamazepine-related T-cell proliferation. Substitution of the terminal amine residue of carbamazepine with a methyl group diminished T-cell proliferation. CONCLUSION: These data show that carbamazepine and certain stable carbamazepine metabolites stimulate T cells rapidly via a direct interaction with MHC and specific T-cell receptors. CLINICAL IMPLICATIONS: Some patients with a history of carbamazepine hypersensitivity possess T cells that cross-react with oxcarbazepine, providing a rationale for cross-sensitivity between the 2 drugs.

Hexose-6-phosphate dehydrogenase modulates 11beta-hydroxysteroid dehydrogenase type 1-dependent metabolism of 7-keto- and 7beta-hydroxy-neurosteroids

BACKGROUND: The role of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in the regulation of energy metabolism and immune system by locally reactivating glucocorticoids has been extensively studied. Experiments determining initial rates of enzyme activity revealed that 11beta-HSD1 can catalyze both the reductase and the dehydrogenase reaction in cell lysates, whereas it predominantly catalyzes the reduction of cortisone to cortisol in intact cells that also express hexose-6-phosphate dehydrogenase (H6PDH), which provides cofactor NADPH. Besides its role in glucocorticoid metabolism, there is evidence that 11beta-HSD1 is involved in the metabolism of 7-keto- and 7-hydroxy-steroids; however the impact of H6PDH on this alternative function of 11beta-HSD1 has not been assessed. METHODOLOGY: We investigated the 11beta-HSD1-dependent metabolism of the neurosteroids 7-keto-, 7alpha-hydroxy- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) and 7-keto- and 7beta-hydroxy-pregnenolone, respectively, in the absence or presence of H6PDH in intact cells. 3D-structural modeling was applied to study the binding of ligands in 11beta-HSD1. PRINCIPAL FINDINGS: We demonstrated that 11beta-HSD1 functions in a reversible way and efficiently catalyzed the interconversion of these 7-keto- and 7-hydroxy-neurosteroids in intact cells. In the presence of H6PDH, 11beta-HSD1 predominantly converted 7-keto-DHEA and 7-ketopregnenolone into their corresponding 7beta-hydroxy metabolites, indicating a role for H6PDH and 11beta-HSD1 in the local generation of 7beta-hydroxy-neurosteroids. 3D-structural modeling offered an explanation for the preferred formation of 7beta-hydroxy-neurosteroids. CONCLUSIONS: Our results from experiments determining the steady state concentrations of glucocorticoids or 7-oxygenated neurosteroids suggested that the equilibrium between cortisone and cortisol and between 7-keto- and 7-hydroxy-neurosteroids is regulated by 11beta-HSD1 and greatly depends on the coexpression with H6PDH. Thus, the impact of H6PDH on 11beta-HSD1 activity has to be considered for understanding both glucocorticoid and neurosteroid action in different tissues.

Proceedings of the Third Biochemical Engineering Symposium (April 28, 1973)

This report presents the proceedings of the Biochemical Engineering Symposium held at Kansas State University, April 28, 1973. Since a number of the contributions will be published in detail elsewhere, only brief summaries of each contribution are included here. Requests for additional information on projects conducted at The University of Nebraska should be directed to Dr. Peter J. Reilly, and those at Kansas State University to the editors. ContentsKenneth J. Jacobson, Andrew H.C. Chan, and Raymond C. Eliason, "Properties and Utilization of Small Particulates in Cattle Manure" Cady R. Engler and James S. Yohn, "Protein from Manure" Robert J. Williams, "Kinetics of Sucrose Inversion Using Invertase Immobilized on Hollow Fibers of Cellulose Acetate" David F. Aldis and Thomas A. Carlisle, "Study of a Triiodide-Resin Complex Disinfection System" John C. Heydweiller, "Modeling and Analysis of Symbiotic Growth" Kenneth J. Jacobson, "Synchronized Growth of the Blue Green Alga Microcystis aeruginosa" Clarence C. Y. Ron arui Lincoln L. S. Yang, "Computer Modeling of the Reductive Pentose Phosphate Cycle" Ming-ching T. Kuo, "Application of a Parallel Biochemical Oxidation Kinetic Model to the Design of an Activated Sludge System Including a Primary Clarifier" Prakash N. Mishra, "Optimal Synthesis of Water Renovation Systems"

Studies of pseudoachondroplasia chondrocytes and knockdown of the cartilage oligomeric matrix protein

Cartilage oligomeric matrix protein (COMP) is a large, homopentameric, extracellular matrix glycoprotein. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EMD1). These dwarfing conditions are caused by retention of misfolded mutant COMP with type IX collagen and matrilin-3 (MATN3) in the rough endoplasmic reticulum (rER) of the chondrocyte. These proteins form a matrix in the rER that continues to expand until it fills the entire cell, eventually causing cell death. Interestingly, loss of COMP in COMP null mice does not affect normal bone development or growth, suggesting that elimination of COMP (wildtype and mutant) expression may prevent PSACH. The hypothesis of these studies was that a hammerhead ribozyme could eliminate or knockdown COMP mRNA expression in PSACH chondrocytes . To test this hypothesis, a human chondrocyte model system that recapitulates the PSACH chondrocyte phenotype was developed by over-expressing mutant (mt-) COMP in normal chondrocytes using a recombinant adenovirus. Chondrocytes over-expressing mt-COMP developed giant rER cisternae containing COMP, type IX collagen and MATN3. Deconvolution microscopy and computer modeling showed that these proteins formed an ordered matrix surrounding a type II pro-collagen core. Additionally, the results show that a hammerhead ribozyme, ribozyme 56 (Ribo56) reduced over-expressed mt-COMP in COS cells and endogenous COMP in normal chondrocytes and mt-COMP in three PSACH chondrocytes cell line (with different mutations) by 40-70%. Altogether, these studies show that the PSACH cellular phenotype can be created in vitro and that the mt-COMP protein burden can be reduced by the presence of a COMP-specific ribozyme. Future studies will focus on designing ribozymes or short interfering RNA (siRNA) technologies that will result in better knockdown of COMP expression as well as the temporal constraints imposed by the PSACH phenotype. ^

Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention

Despite having been identified over thirty years ago and definitively established as having a critical role in driving tumor growth and predicting for resistance to therapy, the KRAS oncogene remains a target in cancer for which there is no effective treatment. KRas is activated b y mutations at a few sites, primarily amino acid substitutions at codon 12 which promote a constitutively active state. I have found that different amino acid substitutions at codon 12 can activate different KRas downstream signaling pathways, determine clonogenic growth potential and determine patient response to molecularly targeted therapies. Computer modeling of the KRas structure shows that different amino acids substituted at the codon 12 position influences how KRas interacts with its effecters. In the absence of a direct inhibitor of mutant KRas several agents have recently entered clinical trials alone and in combination directly targeting two of the common downstream effecter pathways of KRas, namely the Mapk pathway and the Akt pathway. These inhibitors were evaluated for efficacy against different KRAS activating mutations. An isogenic panel of colorectal cells with wild type KRas replaced with KRas G12C, G12D, or G12V at the endogenous loci differed in sensitivity to Mek and Akt inhibition. In contrast, screening was performed in a broad panel of lung cell lines alone and no correlation was seen between types of activating KRAS mutation due to concurrent oncogenic lesions. To find a new method to inhibit KRAS driven tumors, siRNA screens were performed in isogenic lines with and without active KRas. The knockdown of CNKSR1 (CNK1) showed selective growth inhibition in cells with an oncogenic KRAS. The deletion of CNK1 reduces expression of mitotic cell cycle proteins and arrests cells with active KRas in the G1 phase of the cell cycle similar to the deletion of an activated KRas regardless of activating substitution. CNK1 has a PH domain responsible for localizing it to membrane lipids making KRas potentially amenable to inhibition with small molecules. The work has identified a series of small molecules capable of binding to this PH domain and inhibiting CNK1 facilitated KRas signaling.

Mass estimates and morphometric meassurements of Discoaster species and Sphenolithus from Ceara Rise ODP Site 927

Mass estimates for Late Miocene and Pliocene (8.6-3.25 Ma) Discoaster species and Sphenolithus are determined using samples of the equatorial Atlantic (Ceara Rise: ODP Site 927). Based on morphometric measurements, 3D computer models were created for 11 Discoaster species and their volumes calculated. From these, shape factors (ks) were derived to allow calculation of mass for different-sized discoasters and Sphenolithus abies. The mass estimates were then used to calculate the contribution of nannofossils to the total nannofossil carbonate. The discoaster contribution ranges from 10% to 40%, with a decreasing trend through the investigated interval. However, our estimates of total nannofossil carbonate from size-corrected abundance data are consistently 30-50% lower than estimates from grain-size measurement; this suggests that data based on mass estimates need to be interpreted with caution.

Drifting plasma collection by a positive biased tether wire in LEO-like plasma conditions: current measurement and plasma diagnostic

BETs is a three-year project financed by the Space Program of the European Commission, aimed at developing an efficient deorbit system that could be carried on board any future satellite launched into Low Earth Orbit (LEO). The operational system involves a conductive tape-tether left bare to establish anodic contact with the ambient plasma as a giant Langmuir probe. As a part of this project, we are carrying out both numerical and experimental approaches to estimate the collected current by the positive part of the tether. This paper deals with experimental measurements performed in the IONospheric Atmosphere Simulator (JONAS) plasma chamber of the Onera-Space Environment Department. The JONAS facility is a 9- m3 vacuum chamber equipped with a plasma source providing drifting plasma simulating LEO conditions in terms of density and temperature. A thin metallic cylinder, simulating the tether, is set inside the chamber and polarized up to 1000 V. The Earth's magnetic field is neutralized inside the chamber. In a first time, tether collected current versus tether polarization is measured for different plasma source energies and densities. In complement, several types of Langmuir probes are used at the same location to allow the extraction of both ion densities and electron parameters by computer modeling (classical Langmuir probe characteristics are not accurate enough in the present situation). These two measurements permit estimation of the discrepancies between the theoretical collection laws, orbital motion limited law in particular, and the experimental data in LEO-like conditions without magnetic fields. In a second time, the spatial variations and the time evolutions of the plasma properties around the tether are investigated. Spherical and emissive Langmuir probes are also used for a more extensive characterization of the plasma in space and time dependent analysis. Results show the ion depletion because of the wake effect and the accumulation of- ions upstream of the tether. In some regimes (at large positive potential), oscillations are observed on the tether collected current and on Langmuir probe collected current in specific sites.

Metodología de análisis acústico de sitios arqueológicos de Mesoamérica

El presente trabajo busca llenar un vacío existente en cuanto a una metodología general en los estudios Arqueoacústicos para la zona Mesoamericana. El resultado mas importante de ésta tesis es la propuesta de un procedimiento y método para los cuales se detalla el conjunto de operaciones utilizadas en las mediciones particulares involucradas, parámetros y análisis relevantes (de los cuales destacan el decaimiento de energía, tiempos de reverberación, intensidades de sonido directo y total, claridad e inteligibilidad), tipos de simulaciones e incertidumbres y actividades técnicas propias de la Ingeniería Acústica que se proponen para la efectiva y útil integración de la dimensión sonora en el quehacer arqueológico. Una importante consideración de la propuesta metodológica aquí presentada, es la consideración, por un lado, de los resultados obtenidos experimentalmente del trabajo in situ en cada uno de los sitios arqueológicos de interés, y por otro los resultados obtenidos de las simulaciones y modelaciones por computadora de los mismos para su comparación y contraste. La propuesta de sistematización está dividida en tres momentos investigativos, como son la coordinación y trabajo logístico en sitios arqueológicos, su reconocimiento y prospección acústica; el trabajo de campo; y el análisis de los resultados obtenidos. Asimismo, dicha propuesta metodológica, se presenta dividida en tres objetos de estudio fundamentales en el quehacer arqueoacústico: los Fenómenos Sonoros encontrados en sitios arqueológicos; los objetos de generación de sonido, tengan éstos aplicaciones musicales o de otra índole; y finalmente los espacios y recintos arquitectónicos, ya sean cerrados o abiertos, y el estudio de su funcionalidad. El acercamiento a la descripción de cada una de dichas ramas, se realiza mediante la presentación de casos de estudio, para los que se contrastan dos áreas culturales representativas e interrelacionadas de Mesoamérica, la zona del Bajío y la zona Maya. Dicho contraste es llevado a cabo mediante el análisis acústico de 4 zonas arqueológicas que son Plazuelas, Peralta y Cañada de la Virgen, pertenecientes a la zona del Bajio, y Chichen Itzá perteneciente a la zona Maya. De particular importancia para el presente trabajo, es la funcionalidad de los espacios estudiados, específicamente de la de los patios hundidos, característicos de la arquitectura del Bajío, y las plazas públicas que constituyen una estructura integral en muchos sitios arqueológicos, centrales para la comprensión de las conexiones entre el fenómeno sonoro y el comportamiento de una cultura en particular. Asimismo, el análisis de los instrumentos de generación sonora ha permitido realizar inferencias sobre papel del sonido en el comportamiento humano de las culturas en cuestión, completando los modelos acústicos y posibilitando el situar las características de las fuentes sonoras en los espacios resonantes. Los resultados particulares de ésta tesis, han permitido establecer las características acústicas de dichos espacios e instrumentos, así como formular y validar las hipótesis sobre su usos y funciones como espacios para eventos públicos y sociales, así como para representaciones culturales multitudinarios. ABSTRACT The present work, aims to fill a void existing in terms of a general methodology in Archaeoacoustic studies for the Mesoamerican region. The most important result of this thesis is the proposal of a procedure and method for archaeoacoustical studies and its relevant analysis and parameters (of which stand out the energy decay, reverberation times, intensities, clarity and intelligibility), simulations and uncertainty considerations for the effective and useful integration of the sound dimension in the archaeological realm. An important methodological consideration in the present work is the comparison and contrast of the results obtained experimentally on site, and the results of simulations and computer modeling. The proposed systematization is divided into three distinct moments: logistics; fieldwork; and result analysis. Likewise, the presented methodological proposal is divided into three fundamental objects of study: Sound phenomena found in archaeological sites; sound generation objects and instruments; and architectural enclosures and the study of their functionality. Presenting case studies is the approach to the description of each of these branches. Two interrelated Mesoamerican cultural areas are considered, the Bajío and the Mayan. Their contrast is carried out by the acoustic analysis of four archaeological sites: Plazuelas, Peralta and Cañada de la Virgen, belonging to the area of the Bajío, and Chichen Itza belonging to the Maya area. Of particular relevance to this paper, is the acoustic functionality of the public spaces present in many archaeological sites, specifically that of the sunken patios, characteristic of the architecture of the Bajío. The analysis of the sound generation instruments has allowed certain inferences about the role of sound in human behavior of the cultures in question, thus completing the acoustic models. It has also enable the contrast of the characteristics of the sound sources and those of the resonant spaces. The results of this thesis have made it possible to develop and validate certain hypotheses about the uses and functions of certain spaces for public and social events as well as for mass cultural representations.

La aportación de la Península Ibérica a la tecnología naval analizada a través de la interpretación de los documentos y la reproducción de los buques descritos en ellos (1570-1620)

El período de la Historia comprendido entre 1570 y 1620 nos ha dejado un importante conjunto de documentos relacionados con la construcción naval en la Península Ibérica. En una época convulsa en la que los reinos de España y Portugal se aglutinaron bajo una misma Corona, surgen una serie de manuscritos, libros y leyes que reflejan la creciente preocupación de la sociedad por el tema naval. Entre sus páginas encontramos las descripciones del proceso constructivo de los buques que sus autores consideraban más significativos para las demandas que se planteaban en ese momento. Este proceso que combinaba generación de formas y construcción del buque provenía de una secular tradición nacida en el Mediterráneo. Mediante reglas geométricas sencillas, el constructor naval trazaba las secciones centrales y el perfil de la nao, quedando los extremos de la misma (hasta más de la mitad de la eslora) a su buen hacer y experiencia. Las herramientas informáticas de generación de superficies mediante NURBs (Non- Uniform Rational B-spline) permiten reconstruir las formas de los navíos reproduciendo con fiabilidad las carenas de los mismos a partir de los documentos de la época. Mediante un estudio detallado de interpretación de los textos y transcribiendo los procesos, llegamos a obtener con un buen grado de precisión las carenas de los buques descritos en sus páginas. A partir de ahí y mediante el análisis cualitativo y cuantitativo de los parámetros obtenidos es posible valorar si las soluciones representadas por los barcos respondían a las preguntas planteadas por sus autores , la influencia de factores externos a la construcción naval tales como las regulaciones del Estado o identificar su relación con el germen y la expansión de la teoría que ha determinado los efectos de la Ciencia en la Arquitectura Naval. Comenzando por la nao veneciana de 1550, heredera de la secular tradición constructiva mediterránea, hasta llegar a las Reales Ordenanzas promulgadas en 1618, se reproducen hasta nueve carenas a partir de otros tantos documentos, se dibujan sus planos de formas y se exportan para su análisis hidrostático. El trabajo requiere la realización de otros estudios en paralelo necesarios para entender aquellos factores que formaron parte del desarrollo tecnológico naval como son, las unidades de medida en uso en los astilleros, los distintos sistemas de arqueo impuestos por la Corona y la representación de los diferentes instrumentos geométricos de modificación de los parámetros de diseño. A lo largo del trabajo se dan respuesta a interrogantes planteados por la arqueología en relación con el desarrollo de la arquitectura naval poniendo en evidencia que durante este período quedaron establecidos los fundamentos teórico-prácticos de lo que más adelante se convirtió en la ciencia de la ingeniería naval y se plantean nuevos retos para aquellos que deseen continuar la apasionante tarea de la investigación científica de nuestra historia. ABSTRACT The period of the History comprised between 1570 and 1620 has left an important set of shipbuilding documents in the Iberian Peninsula. In a turbulent time in which the kingdoms of Spain and Portugal were ruled under the same Crown, manuscripts, books and laws that reflect the growing concern of society for the naval theme arose. We found among their pages shipbuilding process descriptions of the more relevant vessels that responded to claims that arose at that time. This process brought together hull generation and shipbuilding and came from a secular tradition born in the Mediterranean. By means of simple geometric rules, the shipbuilder traced the central sections and profile of the ship, leaving the ends thereof (almost half of the length) to its good performance and experience. 3D computer modelling software by NURBs (Non-Uniform Rational B-spline) surfaces helps to reconstruct ships hulls from contemporary documents. Through a detailed texts interpretation and transcription processes, we manage to reach with a good degree of accuracy the ship hulls described in its pages. From there and through qualitative and quantitative analysis of the parameters obtained we can assess whether the solutions represented by ships gave response to the questions raised by the authors, the influence of external factors such as shipbuilding state regulations or identify their relationship to the origin and expansion of the theory that has determined the effects of Science in Naval Architecture. From the 1550 Venetian nao, inheritor of the secular Mediterranean building tradition, to the Royal Ordinances enacted in 1618, as nine hulls are reproduced, their line drawings are traced and exported for analysis hydrostatic. Further studies are needed to understand the factors that were part of shipbuilding technology development as the units of measure in use in shipyards, the different official regulations for calculating ship tonnage and the different geometric instruments to amend the design parameters. The work gives response to questions raised by archaeology in relation to the development of naval architecture highlighting that during this period were established the theoretical and practical foundations of what later became the science of naval engineering and raising new challenges for those wishing to continue the exciting task of scientific research of our History.

Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

Effects of unique ion chemistry on thin-film growth by plasma–surface interactions

Plasma processing is a standard industrial method for the modification of material surfaces and the deposition of thin films. Polyatomic ions and neutrals larger than a triatomic play a critical role in plasma-induced surface chemistry, especially in the deposition of polymeric films from fluorocarbon plasmas. In this paper, low energy CF3+ and C3F5+ ions are used to modify a polystyrene surface. Experimental and computational studies are combined to quantify the effect of the unique chemistry and structure of the incident ions on the result of ion-polymer collisions. C3F5+ ions are more effective at growing films than CF3+, both at similar energy/atom of ≈6 eV/atom and similar total kinetic energies of 25 and 50 eV. The composition of the films grown experimentally also varies with both the structure and kinetic energy of the incident ion. Both C3F5+ and CF3+ should be thought of as covalently bound polyatomic precursors or fragments that can react and become incorporated within the polystyrene surface, rather than merely donating F atoms. The size and structure of the ions affect polymer film formation via differing chemical structure, reactivity, sticking probabilities, and energy transfer to the surface. The different reactivity of these two ions with the polymer surface supports the argument that larger species contribute to the deposition of polymeric films from fluorocarbon plasmas. These results indicate that complete understanding and accurate computer modeling of plasma–surface modification requires accurate measurement of the identities, number densities, and kinetic energies of higher mass ions and energetic neutrals.

Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors

The telomerase enzyme is a potential therapeutic target in many human cancers. A series of potent inhibitors has been designed by computer modeling, which exploit the unique structural features of quadruplex DNA. These 3,6,9-trisubstituted acridine inhibitors are predicted to interact selectively with the human DNA quadruplex structure, as a means of specifically inhibiting the action of human telomerase in extending the length of single-stranded telomeric DNA. The anilino substituent at the 9-position of the acridine chromophore is predicted to lie in a third groove of the quadruplex. Calculated relative binding energies predict enhanced selectivity compared with earlier 3,6-disubstituted compounds, as a result of this substituent. The ranking order of energies is in accord with equilibrium binding constants for quadruplex measured by surface plasmon resonance techniques, which also show reduced duplex binding compared with the disubstituted compounds. The 3,6,9-trisubstututed acridines have potent in vitro inhibitory activity against human telomerase, with EC50 values of up to 60 nM.

High-affinity binding sites for histone H1 in plasmid DNA.

The interaction of histone H1 isolated from chicken erythrocytes with restriction fragments from plasmids pBR322 and pUC19 was studied by gel electrophoresis. Certain restriction fragments exhibited unusually high affinity for the histone, forming high molecular mass complexes at protein to DNA ratios at which the other fragments did not show evidence for binding. The highly preferred fragments are intrinsically curved, as judged by their electrophoretic mobility in polyacrylamide gels, by computer modeling, and by imaging with scanning force microscopy. However, control experiments with either curved portions of the same fragments or highly curved kinetoplast DNA fragments showed that the presence of curvature alone was not sufficient for preferential binding. By using various restriction fragments centered around the highly preferred sequence, it was found that the high-affinity binding required in addition the presence of specific sequences on both sides of the region of curvature. Thus, both curvature and the presence of specific sites seem to be required to generate high affinity.

Trees as Tools in Environmental Improvement; An Analysis of the Contribution of Vegetation to Energy and Water Conservation in the Front Range of Colorado

The Denver metropolitan area is facing rapid population growth that increases the stress on already limited resources. Research and advanced computer modeling show that trees, especially those in urban areas, have significant environmental benefits. These benefits include air quality improvements, energy savings, greenhouse gas reduction, and possible water conservation. This Capstone Project applies statistical methods to analyze a small data set of residential homes and their energy and water consumption, as a function of their individual landscape. Results indicate that tree shade can influence water conservation, and that irrigation methods can be an influential factor as well. The Capstone is a preliminary analysis for future study to be performed by the Institute for Environmental Solutions in 2007.