A new analog trigger system for the Cherenkov Telescope array

La astronomía de rayos γ estudia las partículas más energéticas que llegan a la Tierra desde el espacio. Estos rayos γ no se generan mediante procesos térmicos en simples estrellas, sino mediante mecanismos de aceleración de partículas en objetos celestes como núcleos de galaxias activos, púlsares, supernovas, o posibles procesos de aniquilación de materia oscura. Los rayos γ procedentes de estos objetos y sus características proporcionan una valiosa información con la que los científicos tratan de comprender los procesos físicos que ocurren en ellos y desarrollar modelos teóricos que describan su funcionamiento con fidelidad. El problema de observar rayos γ es que son absorbidos por las capas altas de la atmósfera y no llegan a la superficie (de lo contrario, la Tierra será inhabitable). De este modo, sólo hay dos formas de observar rayos γ embarcar detectores en satélites, u observar los efectos secundarios que los rayos γ producen en la atmósfera. Cuando un rayo γ llega a la atmósfera, interacciona con las partículas del aire y genera un par electrón - positrón, con mucha energía. Estas partículas secundarias generan a su vez más partículas secundarias cada vez menos energéticas. Estas partículas, mientras aún tienen energía suficiente para viajar más rápido que la velocidad de la luz en el aire, producen una radiación luminosa azulada conocida como radiación Cherenkov durante unos pocos nanosegundos. Desde la superficie de la Tierra, algunos telescopios especiales, conocidos como telescopios Cherenkov o IACTs (Imaging Atmospheric Cherenkov Telescopes), son capaces de detectar la radiación Cherenkov e incluso de tomar imágenes de la forma de la cascada Cherenkov. A partir de estas imágenes es posible conocer las principales características del rayo γ original, y con suficientes rayos se pueden deducir características importantes del objeto que los emitió, a cientos de años luz de distancia. Sin embargo, detectar cascadas Cherenkov procedentes de rayos γ no es nada fácil. Las cascadas generadas por fotones γ de bajas energías emiten pocos fotones, y durante pocos nanosegundos, y las correspondientes a rayos γ de alta energía, si bien producen más electrones y duran más, son más improbables conforme mayor es su energía. Esto produce dos líneas de desarrollo de telescopios Cherenkov: Para observar cascadas de bajas energías son necesarios grandes reflectores que recuperen muchos fotones de los pocos que tienen estas cascadas. Por el contrario, las cascadas de altas energías se pueden detectar con telescopios pequeños, pero conviene cubrir con ellos una superficie grande en el suelo para aumentar el número de eventos detectados. Con el objetivo de mejorar la sensibilidad de los telescopios Cherenkov actuales, en el rango de energía alto (> 10 TeV), medio (100 GeV - 10 TeV) y bajo (10 GeV - 100 GeV), nació el proyecto CTA (Cherenkov Telescope Array). Este proyecto en el que participan más de 27 países, pretende construir un observatorio en cada hemisferio, cada uno de los cuales contará con 4 telescopios grandes (LSTs), unos 30 medianos (MSTs) y hasta 70 pequeños (SSTs). Con un array así, se conseguirán dos objetivos. En primer lugar, al aumentar drásticamente el área de colección respecto a los IACTs actuales, se detectarán más rayos γ en todos los rangos de energía. En segundo lugar, cuando una misma cascada Cherenkov es observada por varios telescopios a la vez, es posible analizarla con mucha más precisión gracias a las técnicas estereoscópicas. La presente tesis recoge varios desarrollos técnicos realizados como aportación a los telescopios medianos y grandes de CTA, concretamente al sistema de trigger. Al ser las cascadas Cherenkov tan breves, los sistemas que digitalizan y leen los datos de cada píxel tienen que funcionar a frecuencias muy altas (≈1 GHz), lo que hace inviable que funcionen de forma continua, ya que la cantidad de datos guardada será inmanejable. En su lugar, las señales analógicas se muestrean, guardando las muestras analógicas en un buffer circular de unos pocos µs. Mientras las señales se mantienen en el buffer, el sistema de trigger hace un análisis rápido de las señales recibidas, y decide si la imagen que hay en el buér corresponde a una cascada Cherenkov y merece ser guardada, o por el contrario puede ignorarse permitiendo que el buffer se sobreescriba. La decisión de si la imagen merece ser guardada o no, se basa en que las cascadas Cherenkov producen detecciones de fotones en píxeles cercanos y en tiempos muy próximos, a diferencia de los fotones de NSB (night sky background), que llegan aleatoriamente. Para detectar cascadas grandes es suficiente con comprobar que más de un cierto número de píxeles en una región hayan detectado más de un cierto número de fotones en una ventana de tiempo de algunos nanosegundos. Sin embargo, para detectar cascadas pequeñas es más conveniente tener en cuenta cuántos fotones han sido detectados en cada píxel (técnica conocida como sumtrigger). El sistema de trigger desarrollado en esta tesis pretende optimizar la sensibilidad a bajas energías, por lo que suma analógicamente las señales recibidas en cada píxel en una región de trigger y compara el resultado con un umbral directamente expresable en fotones detectados (fotoelectrones). El sistema diseñado permite utilizar regiones de trigger de tamaño seleccionable entre 14, 21 o 28 píxeles (2, 3, o 4 clusters de 7 píxeles cada uno), y con un alto grado de solapamiento entre ellas. De este modo, cualquier exceso de luz en una región compacta de 14, 21 o 28 píxeles es detectado y genera un pulso de trigger. En la versión más básica del sistema de trigger, este pulso se distribuye por toda la cámara de forma que todos los clusters sean leídos al mismo tiempo, independientemente de su posición en la cámara, a través de un delicado sistema de distribución. De este modo, el sistema de trigger guarda una imagen completa de la cámara cada vez que se supera el número de fotones establecido como umbral en una región de trigger. Sin embargo, esta forma de operar tiene dos inconvenientes principales. En primer lugar, la cascada casi siempre ocupa sólo una pequeña zona de la cámara, por lo que se guardan muchos píxeles sin información alguna. Cuando se tienen muchos telescopios como será el caso de CTA, la cantidad de información inútil almacenada por este motivo puede ser muy considerable. Por otro lado, cada trigger supone guardar unos pocos nanosegundos alrededor del instante de disparo. Sin embargo, en el caso de cascadas grandes la duración de las mismas puede ser bastante mayor, perdiéndose parte de la información debido al truncamiento temporal. Para resolver ambos problemas se ha propuesto un esquema de trigger y lectura basado en dos umbrales. El umbral alto decide si hay un evento en la cámara y, en caso positivo, sólo las regiones de trigger que superan el nivel bajo son leídas, durante un tiempo más largo. De este modo se evita guardar información de píxeles vacíos y las imágenes fijas de las cascadas se pueden convertir en pequeños \vídeos" que representen el desarrollo temporal de la cascada. Este nuevo esquema recibe el nombre de COLIBRI (Concept for an Optimized Local Image Building and Readout Infrastructure), y se ha descrito detalladamente en el capítulo 5. Un problema importante que afecta a los esquemas de sumtrigger como el que se presenta en esta tesis es que para sumar adecuadamente las señales provenientes de cada píxel, estas deben tardar lo mismo en llegar al sumador. Los fotomultiplicadores utilizados en cada píxel introducen diferentes retardos que deben compensarse para realizar las sumas adecuadamente. El efecto de estos retardos ha sido estudiado, y se ha desarrollado un sistema para compensarlos. Por último, el siguiente nivel de los sistemas de trigger para distinguir efectivamente las cascadas Cherenkov del NSB consiste en buscar triggers simultáneos (o en tiempos muy próximos) en telescopios vecinos. Con esta función, junto con otras de interfaz entre sistemas, se ha desarrollado un sistema denominado Trigger Interface Board (TIB). Este sistema consta de un módulo que irá montado en la cámara de cada LST o MST, y que estará conectado mediante fibras ópticas a los telescopios vecinos. Cuando un telescopio tiene un trigger local, este se envía a todos los vecinos conectados y viceversa, de modo que cada telescopio sabe si sus vecinos han dado trigger. Una vez compensadas las diferencias de retardo debidas a la propagación en las fibras ópticas y de los propios fotones Cherenkov en el aire dependiendo de la dirección de apuntamiento, se buscan coincidencias, y en el caso de que la condición de trigger se cumpla, se lee la cámara en cuestión, de forma sincronizada con el trigger local. Aunque todo el sistema de trigger es fruto de la colaboración entre varios grupos, fundamentalmente IFAE, CIEMAT, ICC-UB y UCM en España, con la ayuda de grupos franceses y japoneses, el núcleo de esta tesis son el Level 1 y la Trigger Interface Board, que son los dos sistemas en los que que el autor ha sido el ingeniero principal. Por este motivo, en la presente tesis se ha incluido abundante información técnica relativa a estos sistemas. Existen actualmente importantes líneas de desarrollo futuras relativas tanto al trigger de la cámara (implementación en ASICs), como al trigger entre telescopios (trigger topológico), que darán lugar a interesantes mejoras sobre los diseños actuales durante los próximos años, y que con suerte serán de provecho para toda la comunidad científica participante en CTA. ABSTRACT -ray astronomy studies the most energetic particles arriving to the Earth from outer space. This -rays are not generated by thermal processes in mere stars, but by means of particle acceleration mechanisms in astronomical objects such as active galactic nuclei, pulsars, supernovas or as a result of dark matter annihilation processes. The γ rays coming from these objects and their characteristics provide with valuable information to the scientist which try to understand the underlying physical fundamentals of these objects, as well as to develop theoretical models able to describe them accurately. The problem when observing rays is that they are absorbed in the highest layers of the atmosphere, so they don't reach the Earth surface (otherwise the planet would be uninhabitable). Therefore, there are only two possible ways to observe γ rays: by using detectors on-board of satellites, or by observing their secondary effects in the atmosphere. When a γ ray reaches the atmosphere, it interacts with the particles in the air generating a highly energetic electron-positron pair. These secondary particles generate in turn more particles, with less energy each time. While these particles are still energetic enough to travel faster than the speed of light in the air, they produce a bluish radiation known as Cherenkov light during a few nanoseconds. From the Earth surface, some special telescopes known as Cherenkov telescopes or IACTs (Imaging Atmospheric Cherenkov Telescopes), are able to detect the Cherenkov light and even to take images of the Cherenkov showers. From these images it is possible to know the main parameters of the original -ray, and with some -rays it is possible to deduce important characteristics of the emitting object, hundreds of light-years away. However, detecting Cherenkov showers generated by γ rays is not a simple task. The showers generated by low energy -rays contain few photons and last few nanoseconds, while the ones corresponding to high energy -rays, having more photons and lasting more time, are much more unlikely. This results in two clearly differentiated development lines for IACTs: In order to detect low energy showers, big reflectors are required to collect as much photons as possible from the few ones that these showers have. On the contrary, small telescopes are able to detect high energy showers, but a large area in the ground should be covered to increase the number of detected events. With the aim to improve the sensitivity of current Cherenkov showers in the high (> 10 TeV), medium (100 GeV - 10 TeV) and low (10 GeV - 100 GeV) energy ranges, the CTA (Cherenkov Telescope Array) project was created. This project, with more than 27 participating countries, intends to build an observatory in each hemisphere, each one equipped with 4 large size telescopes (LSTs), around 30 middle size telescopes (MSTs) and up to 70 small size telescopes (SSTs). With such an array, two targets would be achieved. First, the drastic increment in the collection area with respect to current IACTs will lead to detect more -rays in all the energy ranges. Secondly, when a Cherenkov shower is observed by several telescopes at the same time, it is possible to analyze it much more accurately thanks to the stereoscopic techniques. The present thesis gathers several technical developments for the trigger system of the medium and large size telescopes of CTA. As the Cherenkov showers are so short, the digitization and readout systems corresponding to each pixel must work at very high frequencies (_ 1 GHz). This makes unfeasible to read data continuously, because the amount of data would be unmanageable. Instead, the analog signals are sampled, storing the analog samples in a temporal ring buffer able to store up to a few _s. While the signals remain in the buffer, the trigger system performs a fast analysis of the signals and decides if the image in the buffer corresponds to a Cherenkov shower and deserves to be stored, or on the contrary it can be ignored allowing the buffer to be overwritten. The decision of saving the image or not, is based on the fact that Cherenkov showers produce photon detections in close pixels during near times, in contrast to the random arrival of the NSB phtotons. Checking if more than a certain number of pixels in a trigger region have detected more than a certain number of photons during a certain time window is enough to detect large showers. However, taking also into account how many photons have been detected in each pixel (sumtrigger technique) is more convenient to optimize the sensitivity to low energy showers. The developed trigger system presented in this thesis intends to optimize the sensitivity to low energy showers, so it performs the analog addition of the signals received in each pixel in the trigger region and compares the sum with a threshold which can be directly expressed as a number of detected photons (photoelectrons). The trigger system allows to select trigger regions of 14, 21, or 28 pixels (2, 3 or 4 clusters with 7 pixels each), and with extensive overlapping. In this way, every light increment inside a compact region of 14, 21 or 28 pixels is detected, and a trigger pulse is generated. In the most basic version of the trigger system, this pulse is just distributed throughout the camera in such a way that all the clusters are read at the same time, independently from their position in the camera, by means of a complex distribution system. Thus, the readout saves a complete camera image whenever the number of photoelectrons set as threshold is exceeded in a trigger region. However, this way of operating has two important drawbacks. First, the shower usually covers only a little part of the camera, so many pixels without relevant information are stored. When there are many telescopes as will be the case of CTA, the amount of useless stored information can be very high. On the other hand, with every trigger only some nanoseconds of information around the trigger time are stored. In the case of large showers, the duration of the shower can be quite larger, loosing information due to the temporal cut. With the aim to solve both limitations, a trigger and readout scheme based on two thresholds has been proposed. The high threshold decides if there is a relevant event in the camera, and in the positive case, only the trigger regions exceeding the low threshold are read, during a longer time. In this way, the information from empty pixels is not stored and the fixed images of the showers become to little \`videos" containing the temporal development of the shower. This new scheme is named COLIBRI (Concept for an Optimized Local Image Building and Readout Infrastructure), and it has been described in depth in chapter 5. An important problem affecting sumtrigger schemes like the one presented in this thesis is that in order to add the signals from each pixel properly, they must arrive at the same time. The photomultipliers used in each pixel introduce different delays which must be compensated to perform the additions properly. The effect of these delays has been analyzed, and a delay compensation system has been developed. The next trigger level consists of looking for simultaneous (or very near in time) triggers in neighbour telescopes. These function, together with others relating to interfacing different systems, have been developed in a system named Trigger Interface Board (TIB). This system is comprised of one module which will be placed inside the LSTs and MSTs cameras, and which will be connected to the neighbour telescopes through optical fibers. When a telescope receives a local trigger, it is resent to all the connected neighbours and vice-versa, so every telescope knows if its neighbours have been triggered. Once compensated the delay differences due to propagation in the optical fibers and in the air depending on the pointing direction, the TIB looks for coincidences, and in the case that the trigger condition is accomplished, the camera is read a fixed time after the local trigger arrived. Despite all the trigger system is the result of the cooperation of several groups, specially IFAE, Ciemat, ICC-UB and UCM in Spain, with some help from french and japanese groups, the Level 1 and the Trigger Interface Board constitute the core of this thesis, as they have been the two systems designed by the author of the thesis. For this reason, a large amount of technical information about these systems has been included. There are important future development lines regarding both the camera trigger (implementation in ASICS) and the stereo trigger (topological trigger), which will produce interesting improvements for the current designs during the following years, being useful for all the scientific community participating in CTA.

The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog

The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] → [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.

Phage display of a catalytic antibody to optimize affinity for transition-state analog binding

Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. “Humanized” 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH → Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.

A thymidine triphosphate shape analog lacking Watson–Crick pairing ability is replicated with high sequence selectivity

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5′-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo− mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F–A pair is associated with a brief pause, whereas that beyond A in the inverted A–F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A–F/F–A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.

Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation

A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. For Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analog N-phosphonacetyl-l-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-Å resolution crystal structure of the C trimer–PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer–PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.

Synthesis and characterization of a novel retinylamine analog inhibitor of constitutively active rhodopsin mutants found in patients with autosomal dominant retinitis pigmentosa

Two different mutations of the active-site Lys-296 in rhodopsin, K296E and K296M, have been found to cause autosomal dominant retinitis pigmentosa (ADRP). In vitro studies have shown that both mutations result in constitutive activation of the protein, suggesting that the activated state of the receptor may be responsible for retinal degeneration in patients with these mutations. Previous work has highlighted the potential of retinylamine analogs as active-site directed inactivators of constitutively active mutants of rhodopsin with the idea that these or related compounds might be used therapeutically for cases of ADRP involving mutations of the active-site Lys. Unfortunately, however, amine derivatives of 11-cis-retinal, although highly effective against a K296G mutant of rhodopsin, were without affect on the two naturally occurring ADRP mutants, presumably because of the greater steric bulk of Glu and Met side chains in comparison to Gly. For this reason we synthesized a retinylamine analog one carbon shorter than the parent 11-cis-retinal and show that this compound is indeed an effective inhibitor of both the K296E and K296M mutants. The 11-cis C19 retinylamine analog 1 inhibits constitutive activation of transducin by these mutants and their constitutive phosphorylation by rhodopsin kinase, and it does so in the presence of continuous illumination from room lights.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} acts as a phosphate analog in proteins phosphorylated on aspartate: Structure of a BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} complex with phosphoserine phosphatase

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

Synthesis and characterization of a photoactivatable analog of corticotropin-releasing factor for specific receptor labeling.

A novel photoactivatable analog of ovine corticotropin-releasing factor (ovine photoCRF) has been synthesized and characterized. A diazirine group, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl residue, was covalently bound to the amino terminus of ovine CRF (oCRF), which was N-terminally extended by a tyrosyl residue for radioactive labeling with 125I. Under mild conditions, photolysis yielded highly reactive carbenes, responsible for the formation of covalent bonds to the CRF receptor. Ovine photoCRF was shown to bind to the high-affinity site of the CRF receptor with a similar Kd value as oCRF. When radioactively iodinated ovine photoCRF (ovine 125I-photoCRF) was covalently linked to rat CRF receptor, type 1 (rCRFR1), permanently transfected into human embryonic kidney (HEK) 293 cells, a highly glycosylated 75-kDa protein was identified with SDS/PAGE. The specificity of ovine 125I-photoCRF was demonstrated by the finding that this analog could be displaced from the receptor by oCRF, but not other unrelated peptides such as vasoactive intestinal peptide. The observed size of the 75-kDa cross-link was in agreement with the molecular weight reported earlier for native CRFR1 from rat brain. Deglycosylation of the 75-kDa cross-link with peptide:N-glycosidase (PNGase) yielded a 46-kDa protein, in agreement with the molecular weight estimated from cDNA coding for rat CRFR1. The developed CRF analog, photoCRF, is expected to facilitate future biochemical and physiological analysis of CRF receptors and--by analogous strategies--of other peptide receptors.

Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060.

The 1,25-dihydroxyvitamin D3 [1,25-(OH)2vitamin D3] analog KH1060 exerts very potent effects on cell proliferation and cell differentiation via the vitamin D receptor (VDR). However, the activities of KH1060 are not associated with an increased affinity for the VDR. We now show that increased stabilization of the VDR-KH1060 complex could be an explanation for its high potencies. VDR half-life studies performed with cycloheximide-translational blocked rat osteoblast-like ROS 17/2.8 cells demonstrated that, in the absence of ligand, VDR levels rapidly decreased. After 2 hr, less than 10% of the initial VDR level could be measured. In the presence of 1,25-(OH)2vitamin D3, the VDR half-life was 15 hr. After 24 hr. less than 20% of the initial VDR content was detectable, whereas, at this time-point, when the cells were incubated with KH1060 80% of the VDR was still present. Differences in 1,25-(OH)2vitamin D3- and KH1060-induced conformational changes of the VDR could underlie the increased VDR stability. As assessed by limited proteolytic digestion analysis, both 1,25-(OH)2vitamin D3 and KH1060 caused a specific conformational change of the VDR. Compared with 1,25-(OH)2vitamin D3, KH1060 induced a conformational change that led to a far more dramatic protection of the VDR against proteolytic degradation. In conclusion, the altered VDR stability and the possibly underlying change in VDR conformation caused by KH1060 could be an explanation for its enhanced bioactivity.

Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.

Design and synthesis of ribonucleic guanidine: a polycationic analog of RNA.

Replacement of the phosphodiester linkages of the polyanion RNA with guanidinium linkers (represented by g) provides the polycation ribonucleic guanidine (RNG). An anticipated structure for the triple-helical hybrid [r(Up)9U.r(Ag)9A.r(Up)9U] is presented. A basic strategy for the synthesis of RNG oligomers is described. Synthetic procedures are provided for tetrameric adenosyl RNG [r(Ag)3A].

Fumonisins and Alternaria alternata lycopersici toxins: sphinganine analog mycotoxins induce apoptosis in monkey kidney cells.

Fusarium moniliforme toxins (fumonisins) and Alternaria alternata lycopersici (AAL) toxins are members of a new class of sphinganine analog mycotoxins that occur widely in the food chain. These mycotoxins represent a serious threat to human and animal health, inducing both cell death and neoplastic events in mammals. The mechanisms by which this family of chemical congeners induce changes in cell homeostasis were investigated in African green monkey kidney cells (CV-1) by assessing the appearance of apoptosis, cell cycle regulation, and putative components of signal transduction pathways involved in apoptosis. Structurally, these mycotoxins resemble the sphingoid bases, sphingosine and sphinganine, that are reported to play critical roles in cell communication and signal transduction. The addition of fumonisin B1 or AAL toxin, TA, to CV-1 cells induced the stereotypical hallmarks of apoptosis, including the formation of DNA ladders, compaction of nuclear DNA, and the subsequent appearance of apoptotic bodies. Neither mycotoxin induced cell death, DNA ladders, or apoptotic bodies in CV-1 cells expressing simian virus 40 large T antigen (COS-7) at toxin concentrations that readily killed CV-1 cells. Fumonisin B1 induced cell cycle arrest in the G1 phase in CV-1 cells but not in COS-7 cells. AAL toxin TA did not arrest cell cycle progression in either cell line. The induction of apoptosis combined with the widespread presence of these compounds in food crops and animal feed identifies a previously unrecognized health risk to humans and livestock. These molecules also represent a new class of natural toxicants that can be used as model compounds to further characterize the molecular and biochemical pathways leading to apoptosis.

Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts.

The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.