NF-kappaB and AP-1 connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.

Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction.

Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46 degrees C, mirroring the finding that adherence was observed in 46 degrees C, but not 37 degrees C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check.

In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z-ring to drive septation. Spatial and temporal control of Z-ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well-studied Min system, less is known about the anti-DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)-mediated NO. SlmA contains a TetR-like DNA-binding fold, and chromatin immunoprecipitation analyses show that SlmA-binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA-FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti-parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA-DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z-ring formation at the correct place and time to effect NO.

Mechanism for sortase localization and the role of sortase localization in efficient pilus assembly in Enterococcus faecalis.

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

Diagnosis of erosive tooth wear.

The clinical diagnosis 'erosion' is made from characteristic deviations from the original anatomical tooth morphology, thus distinguishing acid-induced tissue loss from other forms of wear. Primary pathognomonic features are shallow concavities on smooth surfaces occurring coronal from the enamel-cementum junction. Problems from diagnosing occlusal surfaces and exposed dentine are discussed. Indices for recording erosive wear include morphological as well as quantitative criteria. Currently, various indices are used, each having their virtues and flaws, making the comparison of prevalence studies difficult. The Basic Erosive Wear Examination (BEWE) is described, which is intended to provide an easy tool for research as well as for use in general dental practice. The cumulative score of this index is the sum of the most severe scores obtained from all sextants and is linked to suggestions for clinical management. In addition to recording erosive lesions, the assessment of progression is important as the indication of treatment measures depends on erosion activity. A number of evaluated and sensitive methods for in vitro and in situ approaches are available, but the fundamental problem for their clinical use is the lack of reidentifiable reference areas. Tools for clinical monitoring are described.

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^

OPPL-Galaxy, a Galaxy tool for enhancing ontology exploitation as part of bioinformatics workflows

Biomedical ontologies are key elements for building up the Life Sciences Semantic Web. Reusing and building biomedical ontologies requires flexible and versatile tools to manipulate them efficiently, in particular for enriching their axiomatic content. The Ontology Pre Processor Language (OPPL) is an OWL-based language for automating the changes to be performed in an ontology. OPPL augments the ontologists’ toolbox by providing a more efficient, and less error-prone, mechanism for enriching a biomedical ontology than that obtained by a manual treatment. Results We present OPPL-Galaxy, a wrapper for using OPPL within Galaxy. The functionality delivered by OPPL (i.e. automated ontology manipulation) can be combined with the tools and workflows devised within the Galaxy framework, resulting in an enhancement of OPPL. Use cases are provided in order to demonstrate OPPL-Galaxy’s capability for enriching, modifying and querying biomedical ontologies. Conclusions Coupling OPPL-Galaxy with other bioinformatics tools of the Galaxy framework results in a system that is more than the sum of its parts. OPPL-Galaxy opens a new dimension of analyses and exploitation of biomedical ontologies, including automated reasoning, paving the way towards advanced biological data analyses.

Seismic Risk Scenarios in Puerto Principe (Haiti). A Tool for Reconstruction and Emergency Planning

The 12 January 2010, an earthquake hit the city of Port-au-Prince, capital of Haiti. The earthquake reached a magnitude Mw 7.0 and the epicenter was located near the town of Léogâne, approximately 25 km west of the capital. The earthquake occurred in the boundary region separating the Caribbean plate and the North American plate. This plate boundary is dominated by left-lateral strike slip motion and compression, and accommodates about 20 mm/y slip, with the Caribbean plate moving eastward with respect to the North American plate (DeMets et al., 2000). Initially the location and focal mechanism of the earthquake seemed to involve straightforward accommodation of oblique relative motion between the Caribbean and North American plates along the Enriquillo-Plantain Garden fault system (EPGFZ), however Hayes et al., (2010) combined seismological observations, geologic field data and space geodetic measurements to show that, instead, the rupture process involved slip on multiple faults. Besides, the authors showed that remaining shallow shear strain will be released in future surface-rupturing earthquakes on the EPGFZ. In December 2010, a Spanish cooperation project financed by the Politechnical University of Madrid started with a clear objective: Evaluation of seismic hazard and risk in Haiti and its application to the seismic design, urban planning, emergency and resource management. One of the tasks of the project was devoted to vulnerability assessment of the current building stock and the estimation of seismic risk scenarios. The study was carried out by following the capacity spectrum method as implemented in the software SELENA (Molina et al., 2010). The method requires a detailed classification of the building stock in predominant building typologies (according to the materials in the structure and walls, number of stories and age of construction) and the use of the building (residential, commercial, etc.). Later, the knowledge of the soil characteristics of the city and the simulation of a scenario earthquake will provide the seismic risk scenarios (damaged buildings). The initial results of the study show that one of the highest sources of uncertainties comes from the difficulty of achieving a precise building typologies classification due to the craft construction without any regulations. Also it is observed that although the occurrence of big earthquakes usually helps to decrease the vulnerability of the cities due to the collapse of low quality buildings and the reconstruction of seismically designed buildings, in the case of Port-au-Prince the seismic risk in most of the districts remains high, showing very vulnerable areas. Therefore the local authorities have to drive their efforts towards the quality control of the new buildings, the reinforcement of the existing building stock, the establishment of seismic normatives and the development of emergency planning also through the education of the population.

Un modelo de encaminamiento en las redes móviles ad hoc con QoS

En la última década ha aumentado en gran medida el interés por las redes móviles Ad Hoc. La naturaleza dinámica y sin infraestructura de estas redes, exige un nuevo conjunto de algoritmos y estrategias para proporcionar un servicio de comunicación fiable extremo a extremo. En el contexto de las redes móviles Ad Hoc, el encaminamiento surge como una de las áreas más interesantes para transmitir información desde una fuente hasta un destino, con la calidad de servicio de extremo a extremo. Debido a las restricciones inherentes a las redes móviles, los modelos de encaminamiento tradicionales sobre los que se fundamentan las redes fijas, no son aplicables a las redes móviles Ad Hoc. Como resultado, el encaminamiento en redes móviles Ad Hoc ha gozado de una gran atención durante los últimos años. Esto ha llevado al acrecentamiento de numerosos protocolos de encaminamiento, tratando de cubrir con cada uno de ellos las necesidades de los diferentes tipos de escenarios. En consecuencia, se hace imprescindible estudiar el comportamiento de estos protocolos bajo configuraciones de red variadas, con el fin de ofrecer un mejor encaminamiento respecto a los existentes. El presente trabajo de investigación muestra precisamente una solución de encaminamiento en las redes móviles Ad Hoc. Dicha solución se basa en el mejoramiento de un algoritmo de agrupamiento y la creación de un modelo de encaminamiento; es decir, un modelo que involucra la optimización de un protocolo de enrutamiento apoyado de un mecanismo de agrupación. El algoritmo mejorado, denominado GMWCA (Group Management Weighted Clustering Algorithm) y basado en el WCA (Weighted Clustering Algorithm), permite calcular el mejor número y tamaño de grupos en la red. Con esta mejora se evitan constantes reagrupaciones y que los jefes de clústeres tengan más tiempo de vida intra-clúster y por ende una estabilidad en la comunicación inter-clúster. En la tesis se detallan las ventajas de nuestro algoritmo en relación a otras propuestas bajo WCA. El protocolo de enrutamiento Ad Hoc propuesto, denominado QoS Group Cluster Based Routing Protocol (QoSG-CBRP), utiliza como estrategia el empleo de clúster y jerarquías apoyada en el algoritmo de agrupamiento. Cada clúster tiene un jefe de clúster (JC), quien administra la información de enrutamiento y la envía al destino cuando esta fuera de su área de cobertura. Para evitar que haya constantes reagrupamientos y llamados al algoritmo de agrupamiento se consideró agregarle un jefe de cluster de soporte (JCS), el que asume las funciones del JC, siempre y cuando este haya roto el enlace con los otros nodos comunes del clúster por razones de alejamiento o por desgaste de batería. Matemáticamente y a nivel de algoritmo se han demostrado las mejoras del modelo propuesto, el cual ha involucrado el mejoramiento a nivel de algoritmo de clustering y del protocolo de enrutamiento. El protocolo QoSG-CBRP, se ha implementado en la herramienta de simulación Network Simulator 2 (NS2), con la finalidad de ser comparado con el protocolo de enrutamiento jerárquico Cluster Based Routing Protocol (CBRP) y con un protocolo de enrutamiento Ad Hoc reactivo denominado Ad Hoc On Demand Distance Vector Routing (AODV). Estos protocolos fueron elegidos por ser los que mejor comportamiento presentaron dentro de sus categorías. Además de ofrecer un panorama general de los actuales protocolos de encaminamiento en redes Ad Hoc, este proyecto presenta un procedimiento integral para el análisis de capacidades de la propuesta del nuevo protocolo con respecto a otros, sobre redes que tienen un alto número de nodos. Estas prestaciones se miden en base al concepto de eficiencia de encaminamiento bajo parámetros de calidad de servicio (QoS), permitiendo establecer el camino más corto posible entre un nodo origen y un nodo destino. Con ese fin se han realizado simulaciones con diversos escenarios para responder a los objetivos de la tesis. La conclusiones derivadas del análisis de los resultados permiten evaluar cualitativamente las capacidades que presenta el protocolo dentro del modelo propuesto, al mismo tiempo que avizora un atractivo panorama en líneas futuras de investigación. ABSTRACT In the past decade, the interest in mobile Ad Hoc networks has greatly increased. The dynamic nature of these networks without infrastructure requires a new set of algorithms and strategies to provide a reliable end-to-end communication service. In the context of mobile Ad Hoc networks, routing emerges as one of the most interesting areas for transmitting information from a source to a destination, with the quality of service from end-to-end. Due to the constraints of mobile networks, traditional routing models that are based on fixed networks are not applicable to Ad Hoc mobile networks. As a result, the routing in mobile Ad Hoc networks has experienced great attention in recent years. This has led to the enhancement of many routing protocols, trying to cover with each one of them, the needs of different types of scenarios. Consequently, it is essential to study the behavior of these protocols under various network configurations, in order to provide a better routing scheme. Precisely, the present research shows a routing solution in mobile Ad Hoc networks. This solution is based on the improvement of a clustering algorithm, and the creation of a routing model, ie a model that involves optimizing a routing protocol with the support of a grouping mechanism. The improved algorithm called GMWCA (Group Management Weighted Clustering Algorithm) and based on the WCA (Weighted Clustering Algorithm), allows to calculate the best number and size of groups in the network. With this enhancement, constant regroupings are prevented and cluster heads are living longer intra-cluster lives and therefore stability in inter-cluster communication. The thesis details the advantages of our algorithm in relation to other proposals under WCA. The Ad Hoc routing protocol proposed, called QoS Group Cluster Based Routing Protocol (QoSG-CBRP), uses a cluster-employment strategy and hierarchies supported by the clustering algorithm. Each cluster has a cluster head (JC), who manages the routing information and sends it to the destination when is out of your coverage area. To avoid constant rearrangements and clustering algorithm calls, adding a support cluster head (JCS) was considered. The JCS assumes the role of the JC as long as JC has broken the link with the other nodes in the cluster for common restraining reasons or battery wear. Mathematically and at an algorithm level, the improvements of the proposed model have been showed, this has involved the improvement level clustering algorithm and the routing protocol. QoSG-CBRP protocol has been implemented in the simulation tool Network Simulator 2 (NS2), in order to be compared with the hierarchical routing protocol Cluster Based Routing Protocol (CBRP) and with the reactive routing protocol Ad Hoc On Demand Distance Vector Routing (AODV). These protocols were chosen because they showed the best individual performance in their categories. In addition to providing an overview of existing routing protocols in Ad Hoc networks, this project presents a comprehensive procedure for capacity analysis of the proposed new protocol with respect to others on networks that have a high number of nodes. These benefits are measured based on the concept of routing efficiency under the quality of service (QoS) parameters, thus allowing for the shortest possible path between a source node and a destination node. To meet the objectives of the thesis, simulations have been performed with different scenarios. The conclusions derived from the analysis of the results to assess qualitatively the protocol capabilities presented in the proposed model, while an attractive scenario for future research appears.

Decision-Making Mechanism to Organize the Agenda of a Guide-Robot

One of the major challenges in evolutionary robotics is constituted by the need of the robot being able to make decisions on its own, in accordance with the multiple tasks programmed, optimizing its timings and power. In this paper, we present a new automatic decision making mechanism for a robot guide that allows the robot to make the best choice in order to reach its aims, performing its tasks in an optimal way. The election of which is the best alternative is based on a series of criteria and restrictions of the tasks to perform. The software developed in the project has been verified on the tour-guide robot Urbano. The most important aspect of this proposal is that the design uses learning as the means to optimize the quality in the decision making. The modeling of the quality index of the best choice to perform is made using fuzzy logic and it represents the beliefs of the robot, which continue to evolve in order to match the "external reality”. This fuzzy system is used to select the most appropriate set of tasks to perform during the day. With this tool, the tour guide-robot prepares its agenda daily, which satisfies the objectives and restrictions, and it identifies the best task to perform at each moment. This work is part of the ARABOT project of the Intelligent Control Research Group at the Universidad Politécnica de Madrid to create "awareness" in a robot guide.

Use of infrared thermography as a tool to monitor skin temperature along the recovery process of an anterior cruciate ligament surgery

Infrared thermography (IRT) is a safe and non-invasive tool used for examining physiological functions based on skin temperature (Tsk) control. Thermograms from 25 anterior cruciate ligament (ACL) surgically operated patients (2 female, 23 male) were taken with a FLIR infrared camera according to the protocol established by the International Academy of Clinical Thermology (IACT). This work consists of 4 studies. Studies 1 and 3 were related to establish the probable thermal difference among different moments of an ACL rupture after surgery: before starting the rehabilitation (P0), at the end of rehabilitation (P1) and 18 months from the end of rehabilitation (P2). For this purpose, on the other hand, studies 2 and 4 were related to establish the skin thermal difference (Tsk) between the injured and the non-injured leg in P0, P1 and P2. Results of the first study showed significant temperature increases in the posterior thigh area between P0 and P1 probably due to a compensatory mechanism. According to this, we can conclude that temperature of the posterior area of the injured and noninjured leg has increased from the first to the last day of the rehabilitation process. In the second study we found significant temperature differences between the injured and non-injured leg in both stages of rehabilitation (p<.01). On the one hand, the temperature of the injured leg is higher in the anterior view and the temperature of the non-injured leg is higher in the posterior view. By the time the patients had recovered from the reconstruction, thermal imbalances should have not been shown between symmetrical parts, but differences seemed to be still latent.. Study 3 shows that temperatures seem to be higher after a year and a half (P2) than in P1. Study 4 shows how thermal values 18 months later seemed to be normalized between both legs. No significant differences were found between the injured leg and the noninjured leg after one year and a half of the rehabilitation process. Considering results from Study 3 and 4 we can conclude that patients seemed to have recovered from a thermal point of view. The temperature in P2 was higher but symmetrical. RESUMEN La termografía infrarroja (IRT) es una herramienta segura y no invasiva utilizada para examinar funciones fisiológicas que se basan en el control de temperatura de la piel (Tsk). Termogramas de 25 pacientes intervenidos quirúrgicamente del ligamento cruzado anterior (LCA) (2 mujeres, 23 hombres) fueron tomadas con una cámara de infrarrojos FLIR de acuerdo con el protocolo establecido por la Academia Internacional de Termología Clínica (IACT). Este trabajo consiste en 4 estudios. Los estudios 1 y 3 describen la diferencia térmica entre los diferentes momentos tras la operación del ligamento cruzado anterior: antes de comenzar la rehabilitación (P0), al final de la rehabilitación (P1) y 18 meses tras finalizar la rehabilitación (P2). Por otra parte, los estudios 2 y 4 describen la diferencia de temperatura de la piel (Tsk) entre la pierna lesionada y la pierna no lesionada en P0, P1 y P2. Los resultados del primer estudio mostraron aumentos significativos de temperatura en la zona posterior de los muslos entre P0 y P1, probablemente debido a un mecanismo de compensación. De acuerdo con esto, se puede concluir que la temperatura de la zona posterior de la pierna lesionada y no lesionada se ha incrementado desde el primero hasta el último día del proceso de rehabilitación. En el segundo estudio se encontraron diferencias significativas de temperatura entre la pierna lesionada y no lesionada en ambas etapas de la rehabilitación (p<.01). Por un lado, la temperatura de la pierna lesionada es mayor en la vista anterior. Por otro lado, la temperatura de la pierna no lesionada es mayor en la vista posterior. Una vez que los pacientes se han recuperado de todo el proceso, no deberían existir desequilibrios térmicos entre partes simétricas del cuerpo, pero las diferencias todavía estaban latentes. El tercer estudio muestra que la temperatura es más alta en P2 que en P1. El cuarto estudio muestra cómo los valores térmicos entre ambas piernas en P2 se han normalizado entre ambas piernas. No se encontraron diferencias significativas entre la pierna lesionada y la pierna no lesionada después de 18 meses tras el proceso de rehabilitación. Considerando los resultados del studio 3 y 4, podemos concluir que se ha llegado a la recuperación total desde un punto de vista térmico. La temperatura es más elevada en P2 pero simétrica.

Mechanism-based inhibition of the melatonin rhythm enzyme: Pharmacologic exploitation of active site functional plasticity

Serotonin N-acetyltransferase is the enzyme responsible for the diurnal rhythm of melatonin production in the pineal gland of animals and humans. Inhibitors of this enzyme active in cell culture have not been reported previously. The compound N-bromoacetyltryptamine was shown to be a potent inhibitor of this enzyme in vitro and in a pineal cell culture assay (IC50 ≈ 500 nM). The mechanism of inhibition is suggested to involve a serotonin N-acetyltransferase-catalyzed alkylation reaction between N-bromoacetyltryptamine and reduced CoA, resulting in the production of a tight-binding bisubstrate analog inhibitor. This alkyltransferase activity is apparently catalyzed at a functionally distinct site compared with the acetyltransferase activity active site on serotonin N-acetyltransferase. Such active site plasticity is suggested to result from a subtle conformational alteration in the protein. This plasticity allows for an unusual form of mechanism-based inhibition with multiple turnovers, resulting in “molecular fratricide.” N-bromoacetyltryptamine should serve as a useful tool for dissecting the role of melatonin in circadian rhythm as well as a potential lead compound for therapeutic use in mood and sleep disorders.

On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use an enamine mechanism

Reactive immunization has emerged as a new tool for the study of biological catalysis. A powerful application resulted in catalytic antibodies that use an enamine mechanism akin to that used by the class I aldolases. With regard to the evolution of enzyme mechanisms, we investigated the utility of an enamine pathway for the allylic rearrangement exemplified by Δ5-3-ketosteroid isomerase (KSI; EC 5.3.3.1). Our aldolase antibodies were found to catalyze the isomerization of both steroid model compounds and steroids. The kinetic and chemical studies showed that the antibodies afforded rate accelerations up to a factor of 104 by means of an enamine mechanism in which imine formation was the rate-determining step. In light of our observations and the enzyme studies by other workers, we suggest that an enamine pathway could have been an early, viable KSI mechanism. Although this pathway is amenable to optimization for increased catalytic power, it appears that certain factors precluded its evolution in known KSI enzymes.