Structural and functional analysis of p84N5

Normal development and tissue homeostasis requires the carefully orchestrated balance between cell proliferation and cell death. Cell cycle checkpoints control the extent of cell proliferation. Cell death is coordinated through the activation of a cell suicide pathway that results in the morphologically recognizable form of death, apoptosis. Tumorigenesis requires that the balance between these two pathways be disrupted. The tumor suppressor protein Rb has not only been shown to be involved in the enforcement of cell cycle checkpoints, but has also been implicated in playing a role in the regulation of apoptosis. The manner in which Rb enforces cell cycle checkpoints has been well studied; however, its involvement in the regulation of apoptosis is still very unclear. p84N5 is a novel nuclear death domain containing protein that has been shown to interact with the N-terminus of Rb. The fact that it contains a death domain and the fact that it is nuclear localized possibly provides the first known mechanism for apoptotic signaling from the nucleus. The following study tested the hypothesis that the novel exclusively nuclear death domain containing protein p84N5 is an important mediator of programmed cell death and that its apoptotic function is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. We identified the p84N5 nuclear localization signal (NLS), eliminated it, and tested the functional significance of nuclear localization by using wild type and mutant sequences fused to EGFP-C1 (Clontech) to create wild type GFPN5 and subsequent mutants. The results of these assays demonstrated exclusive nuclear localization of GFPN5 is required for normal p84N5 induced apoptosis. We further conducted large-scale mutagenesis of the GFPN5 construct to identify a minimal region within p84N5 capable of interacting with Rb. We were able to identify a minimal sequence containing p84N5 amino acids 318 to 464 that was capable of interacting with Rb in co-immunoprecipitation assays. We continued by conducting a structural and functional analysis to identify the region or regions within p84N5 responsible for inducing apoptosis. Point mutations and small-scale deletions within the death domain of p84N5 lessened the effect but did not eliminate p84N5-induced cytotoxicity. Further analysis revealed that the minimal sequence of 318 to 464 of p84N5 was capable of inducing apoptosis to a similar degree as wild-type GFPN5 protein. Since amino acids 318 to 464 of p84N5 are capable of inducing apoptosis and interacting with Rb, we propose possible mechanisms whereby p84N5 may function in a Rb regulated manner. These results demonstrate that p84N5 induced apoptosis is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. ^

Perspectives on guidance and social inclusion in a global society

The world is changing rapidly. People today face numerous challenges in achieving a meaningful and fulfilling life. In many countries, there are enormous systemic barriers to address, such as: massive unemployment, HIV/AIDS, social disintegration, and inadequate infrastructure. One job for life is over. For many it never existed. Old metaphors and old models of career development no longer apply. New ways of thinking about careers are necessary, that take into account the context in which people are living, the reality of today's labour market, and the fact people's career-life journey contains many branching paths, barriers, and obstacles, but also allies and sources of assistance. Flexibility is important, as is keeping options open and making sure the journey is meaningful. Guidance professionals need to begin early, working with other professionals and those seeking assistance to develop attitudes that facilitate people taking charge of their own career-life paths. People need a vision for their life that will drive a purposeful approach to career-life planning and avoid floundering. Helping people achieve that direction can be most effectively accomplished when policy makers and practitioners work together to ensure that effective and accessible services are available for those who need them and when a large part of focus in on addressing the context in which marginalized people work and live.

Perspectives on guidance and social inclusion in a global society

The world is changing rapidly. People today face numerous challenges in achieving a meaningful and fulfilling life. In many countries, there are enormous systemic barriers to address, such as: massive unemployment, HIV/AIDS, social disintegration, and inadequate infrastructure. One job for life is over. For many it never existed. Old metaphors and old models of career development no longer apply. New ways of thinking about careers are necessary, that take into account the context in which people are living, the reality of today's labour market, and the fact people's career-life journey contains many branching paths, barriers, and obstacles, but also allies and sources of assistance. Flexibility is important, as is keeping options open and making sure the journey is meaningful. Guidance professionals need to begin early, working with other professionals and those seeking assistance to develop attitudes that facilitate people taking charge of their own career-life paths. People need a vision for their life that will drive a purposeful approach to career-life planning and avoid floundering. Helping people achieve that direction can be most effectively accomplished when policy makers and practitioners work together to ensure that effective and accessible services are available for those who need them and when a large part of focus in on addressing the context in which marginalized people work and live.

Perspectives on guidance and social inclusion in a global society

The world is changing rapidly. People today face numerous challenges in achieving a meaningful and fulfilling life. In many countries, there are enormous systemic barriers to address, such as: massive unemployment, HIV/AIDS, social disintegration, and inadequate infrastructure. One job for life is over. For many it never existed. Old metaphors and old models of career development no longer apply. New ways of thinking about careers are necessary, that take into account the context in which people are living, the reality of today's labour market, and the fact people's career-life journey contains many branching paths, barriers, and obstacles, but also allies and sources of assistance. Flexibility is important, as is keeping options open and making sure the journey is meaningful. Guidance professionals need to begin early, working with other professionals and those seeking assistance to develop attitudes that facilitate people taking charge of their own career-life paths. People need a vision for their life that will drive a purposeful approach to career-life planning and avoid floundering. Helping people achieve that direction can be most effectively accomplished when policy makers and practitioners work together to ensure that effective and accessible services are available for those who need them and when a large part of focus in on addressing the context in which marginalized people work and live.

A Stress-Detection System Based on Physiological Signals and Fuzzy Logic

A stress-detection system is proposed based on physiological signals. Concretely, galvanic skin response (GSR) and heart rate (HR) are proposed to provide information on the state of mind of an individual, due to their nonintrusiveness and noninvasiveness. Furthermore, specific psychological experiments were designed to induce properly stress on individuals in order to acquire a database for training, validating, and testing the proposed system. Such system is based on fuzzy logic, and it described the behavior of an individual under stressing stimuli in terms of HR and GSR. The stress-detection accuracy obtained is 99.5% by acquiring HR and GSR during a period of 10 s, and what is more, rates over 90% of success are achieved by decreasing that acquisition period to 3-5 s. Finally, this paper comes up with a proposal that an accurate stress detection only requires two physiological signals, namely, HR and GSR, and the fact that the proposed stress-detection system is suitable for real-time applications.

Rapid prototyping for biomedical engineering: current capabilities and Challenges

A new set of manufacturing technologies has emerged in the past decades to address market requirements in a customized way and to provide support for research tasks that require prototypes. These new techniques and technologies are usually referred to as rapid prototyping and manufacturing technologies, and they allow prototypes to be produced in a wide range of materials with remarkable precision in a couple of hours. Although they have been rapidly incorporated into product development methodologies, they are still under development, and their applications in bioengineering are continuously evolving. Rapid prototyping and manufacturing technologies can be of assistance in every stage of the development process of novel biodevices, to address various problems that can arise in the devices' interactions with biological systems and the fact that the design decisions must be tested carefully. This review focuses on the main fields of application for rapid prototyping in biomedical engineering and health sciences, as well as on the most remarkable challenges and research trends.

Advanced system of planning and organization of classes at the university

This document is the result of a process of web development to create a tool that will allow to Cracow University of Technology consult, create and manage timetables. The technologies chosen for this purpose are Apache Tomcat Server, My SQL Community Server, JDBC driver, Java Servlets and JSPs for the server side. The client part counts on Javascript, jQuery, AJAX and CSS technologies to perform the dynamism. The document will justify the choice of these technologies and will explain some development tools that help in the integration and development of all this elements: specifically, NetBeans IDE and MySQL workbench have been used as helpful tools. After explaining all the elements involved in the development of the web application, the architecture and the code developed are explained through UML diagrams. Some implementation details related to security are also deeper explained through sequence diagrams. As the source code of the application is provided, an installation manual has been developed to run the project. In addition, as the platform is intended to be a beta that will be grown, some unimplemented ideas for future development are also exposed. Finally, some annexes with important files and scripts related to the initiation of the platform are attached. This project started through an existing tool that needed to be expanded. The main purpose of the project along its development has focused on setting the roots for a whole new platform that will replace the existing one. For this goal, it has been needed to make a deep inspection on the existing web technologies: a web server and a SQL database had to be chosen. Although the alternatives were a lot, Java technology for the server was finally selected because of the big community backwards, the easiness of modelling the language through UML diagrams and the fact of being free license software. Apache Tomcat is the open source server that can use Java Servlet and JSP technology. Related to the SQL database, MySQL Community Server is the most popular open-source SQL Server, with a big community after and quite a lot of tools to manage the server. JDBC is the driver needed to put in contact Java and MySQL. Once we chose the technologies that would be part of the platform, the development process started. After a detailed explanation of the development environment installation, we used UML use case diagrams to set the main tasks of the platform; UML class diagrams served to establish the existing relations between the classes generated; the architecture of the platform was represented through UML deployment diagrams; and Enhanced entity–relationship (EER) model were used to define the tables of the database and their relationships. Apart from the previous diagrams, some implementation issues were explained to make a better understanding of the developed code - UML sequence diagrams helped to explain this. Once the whole platform was properly defined and developed, the performance of the application has been shown: it has been proved that with the current state of the code, the platform covers the use cases that were set as the main target. Nevertheless, some requisites needed for the proper working of the platform have been specified. As the project is aimed to be grown, some ideas that could not be added to this beta have been explained in order not to be missed for future development. Finally, some annexes containing important configuration issues for the platform have been added after proper explanation, as well as an installation guide that will let a new developer get the project ready. In addition to this document some other files related to the project are provided: - Javadoc. The Javadoc containing the information of every Java class created is necessary for a better understanding of the source code. - database_model.mwb. This file contains the model of the database for MySQL Workbench. This model allows, among other things, generate the MySQL script for the creation of the tables. - ScheduleManager.war. The WAR file that will allow loading the developed application into Tomcat Server without using NetBeans. - ScheduleManager.zip. The source code exported from NetBeans project containing all Java packages, JSPs, Javascript files and CSS files that are part of the platform. - config.properties. The configuration file to properly get the names and credentials to use the database, also explained in Annex II. Example of config.properties file. - db_init_script.sql. The SQL query to initiate the database explained in Annex III. SQL statements for MySQL initialization. RESUMEN. Este proyecto tiene como punto de partida la necesidad de evolución de una herramienta web existente. El propósito principal del proyecto durante su desarrollo se ha centrado en establecer las bases de una completamente nueva plataforma que reemplazará a la existente. Para lograr esto, ha sido necesario realizar una profunda inspección en las tecnologías web existentes: un servidor web y una base de datos SQL debían ser elegidos. Aunque existen muchas alternativas, la tecnología Java ha resultado ser elegida debido a la gran comunidad de desarrolladores que tiene detrás, además de la facilidad que proporciona este lenguaje a la hora de modelarlo usando diagramas UML. Tampoco hay que olvidar que es una tecnología de uso libre de licencia. Apache Tomcat es el servidor de código libre que permite emplear Java Servlets y JSPs para hacer uso de la tecnología de Java. Respecto a la base de datos SQL, el servidor más popular de código libre es MySQL, y cuenta también con una gran comunidad detrás y buenas herramientas de modelado, creación y gestión de la bases de datos. JDBC es el driver que va a permitir comunicar las aplicaciones Java con MySQL. Tras elegir las tecnologías que formarían parte de esta nueva plataforma, el proceso de desarrollo tiene comienzo. Tras una extensa explicación de la instalación del entorno de desarrollo, se han usado diagramas de caso de UML para establecer cuáles son los objetivos principales de la plataforma; los diagramas de clases nos permiten realizar una organización del código java desarrollado de modo que sean fácilmente entendibles las relaciones entre las diferentes clases. La arquitectura de la plataforma queda definida a través de diagramas de despliegue. Por último, diagramas EER van a definir las relaciones entre las tablas creadas en la base de datos. Aparte de estos diagramas, algunos detalles de implementación se van a justificar para tener una mejor comprensión del código desarrollado. Diagramas de secuencia ayudarán en estas explicaciones. Una vez que toda la plataforma haya quedad debidamente definida y desarrollada, se va a realizar una demostración de la misma: se demostrará cómo los objetivos generales han sido alcanzados con el desarrollo actual del proyecto. No obstante, algunos requisitos han sido aclarados para que la plataforma trabaje adecuadamente. Como la intención del proyecto es crecer (no es una versión final), algunas ideas que se han podido llevar acabo han quedado descritas de manera que no se pierdan. Por último, algunos anexos que contienen información importante acerca de la plataforma se han añadido tras la correspondiente explicación de su utilidad, así como una guía de instalación que va a permitir a un nuevo desarrollador tener el proyecto preparado. Junto a este documento, ficheros conteniendo el proyecto desarrollado quedan adjuntos. Estos ficheros son: - Documentación Javadoc. Contiene la información de las clases Java que han sido creadas. - database_model.mwb. Este fichero contiene el modelo de la base de datos para MySQL Workbench. Esto permite, entre otras cosas, generar el script de iniciación de la base de datos para la creación de las tablas. - ScheduleManager.war. El fichero WAR que permite desplegar la plataforma en un servidor Apache Tomcat. - ScheduleManager.zip. El código fuente exportado directamente del proyecto de Netbeans. Contiene todos los paquetes de Java generados, ficheros JSPs, Javascript y CSS que forman parte de la plataforma. - config.properties. Ejemplo del fichero de configuración que permite obtener los nombres de la base de datos - db_init_script.sql. Las consultas SQL necesarias para la creación de la base de datos.

Identification and characterization of amelogenin genes in monotremes, reptiles, and amphibians

Two features make the tooth an excellent model in the study of evolutionary innovations: the relative simplicity of its structure and the fact that the major tooth-forming genes have been identified in eutherian mammals. To understand the nature of the innovation at the molecular level, it is necessary to identify the homologs of tooth-forming genes in other vertebrates. As a first step toward this goal, homologs of the eutherian amelogenin gene have been cloned and characterized in selected species of monotremes (platypus and echidna), reptiles (caiman), and amphibians (African clawed toad). Comparisons of the homologs reveal that the amelogenin gene evolves quickly in the repeat region, in which numerous insertions and deletions have obliterated any similarity among the genes, and slowly in other regions. The gene organization, the distribution of hydrophobic and hydrophilic segments in the encoded protein, and several other features have been conserved throughout the evolution of the tetrapod amelogenin gene. Clones corresponding to one locus only were found in caiman, whereas the clawed toad possesses at least two amelogenin-encoding loci.

Cloning and characterization of TDD5, an androgen target gene that is differentially repressed by testosterone and dihydrotestosterone

By using mRNA polymerase chain reaction differential display technique (DDPCR), we have identified one early responsive cDNA fragment, TDD5, from a 5α-reductase-deficient T cell hybridoma. The DDPCR profiles of TDD5 suggest that its expression can be repressed by testosterone (T) within 2 hr. More importantly, both DDPCR and Northern blot analysis further demonstrated that the expression of TDD5 was differentially repressed by T and dihydrotestosterone (DHT) at the mRNA level. To our knowledge, this is the first androgen target gene to show a preference in response to T over DHT in cell culture. TDD5 is expressed in several tissues with particular abundance in kidney. Full-length TDD5 cDNA (2,916 bp) encodes a protein with a calculated molecular weight of 42,000. Finally, our animal studies further confirm that TDD5 mRNA levels can be repressed to the basal level 8 hr after DHT administration. The isolation and characterization of the early-responsive androgen target gene TDD5 and the fact that TDD5 mRNA level can be differentially regulated by T and DHT may provide a useful tool to study the molecular mechanism of androgen preference on target gene regulation.

Dissociation between bone resorption and bone formation in osteopenic transgenic mice

Bone mass is maintained constant in vertebrates through bone remodeling (BR). BR is characterized by osteoclastic resorption of preexisting bone followed by de novo bone formation by osteoblasts. This sequence of events and the fact that bone mass remains constant in physiological situation lead to the assumption that resorption and formation are regulated by each other during BR. Recent evidence shows that cells of the osteoblastic lineage are involved in osteoclast differentiation. However, the existence of a functional link between the two activities, formation and resorption, has never been shown in vivo. To define the role of bone formation in the control of bone resorption, we generated an inducible osteoblast ablation mouse model. These mice developed a reversible osteopenia. Functional analyses showed that in the absence of bone formation, bone resorption continued to occur normally, leading to an osteoporosis of controllable severity, whose appearance could be prevented by an antiresorptive agent. This study establishes that bone formation and/or bone mass do not control the extent of bone resorption in vivo.

Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds

The promoters of MEA (FIS1), FIS2, and FIE (FIS3), genes that repress seed development in the absence of pollination, were fused to β-glucuronidase (GUS) to study their activity pattern. The FIS2∷GUS product is found in the embryo sac, in each of the polar cell nuclei, and in the central cell nucleus. After pollination, the maternally derived FIS2∷GUS protein occurs in the nuclei of the cenocytic endosperm. Before cellularization of the endosperm, activity is terminated in the micropylar and central nuclei of the endosperm and subsequently in the nuclei of the chalazal cyst. MEA∷GUS has a pattern of activity similar to that of FIS2∷GUS, but FIE∷GUS protein is found in many tissues, including the prepollination embryo sac, and in embryo and endosperm postpollination. The similarity in mutant phenotypes; the activity of FIE, MEA, and FIS2 in the same cells in the embryo sac; and the fact that MEA and FIE proteins interact in a yeast two-hybrid system suggest that these proteins operate in the same system of control of seed development. Maternal and not paternal FIS2∷GUS, MEA∷GUS, and FIE∷GUS show activity in early endosperm, so these genes may be imprinted. When fis2, mea, and fie mutants are pollinated, seed development is arrested at the heart embryo stage. The seed arrest of mea and fis2 is avoided when they are fertilized by a low methylation parent. The wild-type alleles of MEA or FIS2 are not required. The parent-of-origin-determined differential activity of MEA, FIS2, and FIE is not dependent on DNA methylation, but methylation does control some gene(s) that have key roles in seed development.

Sodium channels and pain

Although it is well established that hyperexcitability and/or increased baseline sensitivity of primary sensory neurons can lead to abnormal burst activity associated with pain, the underlying molecular mechanisms are not fully understood. Early studies demonstrated that, after injury to their axons, neurons can display changes in excitability, suggesting increased sodium channel expression, and, in fact, abnormal sodium channel accumulation has been observed at the tips of injured axons. We have used an ensemble of molecular, electrophysiological, and pharmacological techniques to ask: what types of sodium channels underlie hyperexcitability of primary sensory neurons after injury? Our studies demonstrate that multiple sodium channels, with distinct electrophysiological properties, are encoded by distinct mRNAs within small dorsal root ganglion (DRG) neurons, which include nociceptive cells. Moreover, several DRG neuron-specific sodium channels now have been cloned and sequenced. After injury to the axons of DRG neurons, there is a dramatic change in sodium channel expression in these cells, with down-regulation of some sodium channel genes and up-regulation of another, previously silent sodium channel gene. This plasticity in sodium channel gene expression is accompanied by electrophysiological changes that poise these cells to fire spontaneously or at inappropriate high frequencies. Changes in sodium channel gene expression also are observed in experimental models of inflammatory pain. Thus, sodium channel expression in DRG neurons is dynamic, changing significantly after injury. Sodium channels within primary sensory neurons may play an important role in the pathophysiology of pain.

A total system approach to sustainable pest management

A fundamental shift to a total system approach for crop protection is urgently needed to resolve escalating economic and environmental consequences of combating agricultural pests. Pest management strategies have long been dominated by quests for “silver bullet” products to control pest outbreaks. However, managing undesired variables in ecosystems is similar to that for other systems, including the human body and social orders. Experience in these fields substantiates the fact that therapeutic interventions into any system are effective only for short term relief because these externalities are soon “neutralized” by countermoves within the system. Long term resolutions can be achieved only by restructuring and managing these systems in ways that maximize the array of “built-in” preventive strengths, with therapeutic tactics serving strictly as backups to these natural regulators. To date, we have failed to incorporate this basic principle into the mainstream of pest management science and continue to regress into a foot race with nature. In this report, we establish why a total system approach is essential as the guiding premise of pest management and provide arguments as to how earlier attempts for change and current mainstream initiatives generally fail to follow this principle. We then draw on emerging knowledge about multitrophic level interactions and other specific findings about management of ecosystems to propose a pivotal redirection of pest management strategies that would honor this principle and, thus, be sustainable. Finally, we discuss the potential immense benefits of such a central shift in pest management philosophy.

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.

Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons.

The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-dependent PDE family designated as PDE1C. This enzyme shows high affinity for cAMP and cGMP, having a Km for cAMP much lower than that of any other neuronal Ca2+/calmodulin-dependent PDE. The mRNA encoding this enzyme is highly enriched in olfactory epithelium and is not detected in six other tissues tested. However, RNase protection analyses indicate that other alternative splice variants related to this enzyme are expressed in several other tissues. Within the olfactory epithelium, this enzyme appears to be expressed exclusively in the sensory neurons. The high affinity for cAMP of this Ca2+/calmodulin-dependent PDE and the fact that its mRNA is highly concentrated in olfactory sensory neurons suggest an important role for it in a Ca(2+)-regulated olfactory signal termination.