Results of students surveys in similar courses given in different centers of the Technical University of Madrid

We present and analyze the results of surveys conducted in recent years with students from two related subjects, but taught in different centers of the University of Madrid. These surveys are part of the objectives of various projects of educational innovation, and applied through the platform Moodle.

Development of a model of cross-curricular competencies of students at the Technical University of Madrid

It is known that cross-curricular competences are required for main companies all over the world to be part of our university graduates as technical knowledge does. That is the reason which has led the university structure to include these competences in the every degree curriculo validated since the European Higher Education Area (EHEA)was introduced in the Spanish university context. But the way used for incorporating them has been developed without the necessary guidelines to generate a qualified model.

CAS: A tool for improving autonomous work

The EHEA proposes a student-centered teaching model. Therefore, it seems necessary to actively involve the students in the teaching-learning process. Increasing the active participation of the students is not always easy in mathematical topics, since, when the students just enter the University, their ability to carry out autonomous mathematical work is scarce. In this paper we present some experiences related with the use of Computer Algebra Systems (CAS). All the experiences are designed in order to develop some mathematical competencies and mainly self-learning, the use of technology and team-work. The experiences include some teachers? proposals including: small projects to be executed in small groups, participation in competitions, the design of different CAS-Toolboxes, etc. The results obtained in the experiences, carried out with different groups of students from different engineering studies at different universities, makes us slightly optimistic about the educational value of the model.

"Deporte inclusivo en la escuela": diseño y análisis de un programa de intervencion para promover la inclusión del alumnado con discapacidad en Educación Física

La Educación Física se muestra, a priori, como un área idónea para trabajar intervenciones educativas para facilitar la inclusión de alumnos con discapacidad. Sin embargo, poco se conoce sobre el potencial que presentan los deportes adaptados y paralímpicos como contenido para fomentar la sensibilización y concienciación del alumnado sin discapacidad, especialmente en situaciones inclusivas de práctica. Por otro lado, la actitud del docente y su formación se presentan como claves en este contexto, ya que es quien en última instancia selecciona los contenidos a trabajar. Entendemos que en EF disponemos de una ocasión única en el curriculum para fomentar la participación activa y efectiva del alumno con discapacidad en clase (especialmente a nivel de desarrollo de la competencia motriz, entre otras), si bien esto depende de factores relacionados con los dos agentes anteriores. Es por todo ello que la Educación Física, como protagonista y como contenido, se muestra como un contexto adecuado para la investigación de los procesos de inclusión de alumnos con discapacidad en el ámbito educativo. Este trabajo de investigación pretende arrojar luz a los interrogantes que condicionan y limitan este contexto, desde una perspectiva multidisciplinar, con distintas metodologías, sobre los tres agentes indicados. La falta de consenso en la literatura en cuanto a las características y tipo de intervenciones eficaces para facilitar esta sensibilización del alumnado, unido a que es un ámbito relativamente reciente como tema de investigación, nos ha impulsado a trabajar en esta línea. El primer objetivo de este trabajo de investigación fue diseñar e implementar un programa de sensibilización y concienciación hacia la discapacidad basado en los deportes adaptados en el área de Educación Física para alumnos de secundaria y bachillerato. Inicialmente, se realizó una búsqueda bibliográfica tanto a nivel nacional como internacional, con el fin de definir las características principales que aporta la literatura científica en este aspecto. Apoyándonos por un lado, en el programa educativo Paralympic School Day (CPI, 2004) y por otro, en la citada revisión, desarrollamos un planteamiento inicial de estructura y fases. Dicho proyecto, fue presentado al Comité Paralímpico Español y a las federaciones deportivas españolas para personas con discapacidad, con la finalidad de recabar su apoyo institucional en forma de aval y recursos no solo a nivel económico sino también como apoyo logístico y de difusión. Tras su aprobación y gracias también al apoyo de la UPM, la Fundación Sanitas y Liberty Seguros, se procedió a diseñar el programa. Para el desarrollo de los materiales didácticos se contactó con expertos en la materia de EF y Actividad Física Adaptada tanto del ámbito educativo (profesores de educación secundaria y profesorado universitario) como del deportivo a nivel nacional. A su vez, se comenzó a difundir entre el profesorado de los centros con el fin de detectar su interés en participar durante el curso académico (2012-2013) en el programa “Deporte Inclusivo en la Escuela”. Con la finalización del desarrollo de los materiales didácticos, se visitó a los centros educativos para presentar el dossier informativo donde se explicaba el programa, así como las características y fases para su implementación. El programa está fundamentado en la Teoría del Contacto (Allport, 1954) y basado en los deportes adaptados y paralímpicos, planteado con una metodología inclusiva, seleccionando la información, la simulación y el contacto directo como estrategias para el fomento de la sensibilización y concienciación hacia la inclusión. En la reunión celebrada en la Facultad de Ciencias de la Actividad Física y del Deporte (INEF-UPM) en febrero de 2013, se coordinó junto con el profesorado la implementación del programa en cada uno de los 13 centros educativos, con acciones concretas como la adecuación de la propuesta didáctica en la planificación anual del profesor, el préstamo de material o la ponencia del deportista paralímpico entre otras cuestiones. Para la consecución del objetivo 2 de este trabajo, analizar el efecto del programa en los distintos agentes implicados en el mismo, alumnos sin discapacidad, profesorado de EF y alumnos con discapacidad, se calendarizó la toma de datos y la administración de las diferentes herramientas metodológicas para antes (pretest) como después de la intervención (posttets). En el caso de los alumnos sin discapacidad (N= 1068), se analizó el efecto de la intervención sobre la actitud hacia la inclusión, utilizando el cuestionario “Children Attitude Integrated Physical Education-Revised” (CAIPE-R; Block, 1995) de carácter cuantitativo tras su validación y adaptación al contexto español. Los resultados mostraron cambios significativos positivos en la actitud el grupo que mantuvo un contacto no estructurado con alumnos con discapacidad. En esta muestra también se midió la actitud hacia el juego cooperativo con compañeros con discapacidad en clases de EF usando el cuestionario “Children's Beliefs Toward Cooperative Playing With Peers With Disabilities in Physical Education” (CBIPPD-MPE; Obrusnikova, Block, y Dillon, 2010). El desarrollo de un sistema de categorías fundamentado en la Teoría del Comportamiento Planificado (Azjen, 1991) sirvió como base para el análisis de las creencias del alumnado sin discapacidad. Tras la intervención, las creencias conductuales emergentes se mantuvieron, excepto en el caso de los factores identificados como obstáculos de la inclusión. En el caso de las creencias normativas también se mantuvieron tras la intervención y respecto a las creencias de control, los alumnos identificaron al profesor como principal agente facilitador de la inclusión. En el caso de los profesores de EF participantes en el programa (N=18), se analizó el efecto del programa en su actitud hacia la inclusión de alumnos con discapacidad en EF con el cuestionario “Attitud toward inclusion of individual with physical disabilities in Physical Education” (ATISDPE-R; Kudláèek, Válková, Sherrill, Myers, y French, 2002). Los resultados mostraron que no se produjeron diferencias significativas tras la intervención en la actitud general, encontrando algunas diferencias en determinados ítems relacionados con los beneficios de la inclusión en los alumnos sin discapacidad relacionados con los docentes con experiencia previa con discapacidad y en EF antes de la intervención. La otra dimensión analizada fue el efecto de la intervención en la autoeficacia del profesor en la enseñanza de la EF en condiciones inclusivas, habiendo utilizado el cuestionario "Self-efficacy in teaching PE under inclusive conditions" (SEIPE; Hutzler, Zach, y Gafni, 2005). Los resultados en este caso muestran diferencias significativas positivas en cuestiones relacionadas como sentirse capaces de mejorar las condiciones óptimas de enseñanza con alumnos con discapacidad física como movilidad reducida severa y amputación y discapacidad visual tanto en situaciones deportivas, como juegos o actividades fuera del centro educativo a favor de los docentes. En cuanto al género, los hombres obtuvieron valores superiores a las mujeres en relación a sentirse más capaces de incluir a alumnos con discapacidad física tanto en juegos durante el recreo como en la enseñanza de técnica deportiva. Los profesores con menos de 10 años de docencia mostraron valores más positivos en cuanto a sentirse capaces de incluir a un alumno con discapacidad fisica en deportes durante su tiempo libre. El análisis del diario del profesor muestra por un lado, las tendencias emergentes como principales elementos facilitadores u obstaculizadores de la inclusión en EF, identificando al propio alumno sin discapacidad, el propio profesor, los contenidos, los materiales y la organización. Por otro lado, el análisis de los contenidos propuestos en el programa. En el caso de los alumnos con discapacidad (N=22), se analizó el impacto del programa de intervención en el autoconcepto, con el cuestionario "Autoconcepto forma 5" (AF5; F. García y Musitu, 2001). Se encontraron diferencias significativas a favor de las mujeres antes de la intervención en la dimensión familiar, mientras que los hombres obtuvieron valores más altos en las dimensiones social y físico. En cuanto a la edad, se encontraron diferencias significativas antes de la intervención, con valores superiores en los alumnos más jóvenes (12-14 años) en la dimensión físico, mientras que los alumnos mayores (15-17 años) mostraron valores más altos en la dimensión social del cuestionario. Respeto al tipo de discapacidad, los alumnos con discapacidad motórica mostraron mejores valores que los que tienen discapacidad auditiva para la dimensión físico antes de la intervención. En cuanto al autoconcepto general, las diferencias significativas positivas se producen en la dimensión académica. En cuanto al efecto del programa en la autoestima de los alumnos con discapacidad, se utilizó la Escala de "Autoestima de Rosenberg" (Rosenberg, 1989), no obteniendo diferencias significativas en cuanto el género. Apareciendo diferencias significativas antes de la intervención en el caso de la variable edad en los alumnos más jóvenes, en cuanto a que desearían valorarse más, y en los alumnos con discapacidad auditiva en que no se sienten muy orgullosos de ellos mismos. Se produce una mejora en la autoestima general en cuanto a que se sienten menos inútiles tras la intervención. En relación al objetivo 3 de este trabajo, tras el análisis de los resultados y haber discutido los mismos con los autores de referencia, emergió la propuesta de orientaciones tanto para los programa de intervención en EF para la sensibilización y concienciación del alumnado hacia la inclusión como de cara a la formación específica del profesorado, como clave en este tipo de intervenciones. Creemos que el programa “Deporte Inclusivo Escuela” se convierte en un elemento transformador de la realidad, ya que responde a las necesidades detectadas a la luz de esta investigación y que vienen a dar respuesta a los distintos agentes implicados en su desarrollo. Por un lado, atiende la demanda del ámbito educativo en cuanto a las necesidades de formación del profesorado, sensibilización y concienciación del alumnado sin discapacidad, además de facilitar oportunidades de participación activa al alumno con discapacidad en las sesiones de EF. Por otro lado, satisface la demanda por parte de las instituciones deportivas del ámbito de la discapacidad en cuanto a la promoción y difusión de los deportes adaptados y paralímpicos. Por último, desde el ámbito universitario, se muestra como un recurso en la formación del alumnado del grado en Ciencias de la Actividad Física y del Deporte, por su participación directa con la discapacidad. Por estos motivos, este trabajo se muestra como un punto de partida adecuado para seguir avanzando en la investigación en esta área. ABSTRACT Physical Education (PE) seems a priori, as a suitable area to work educational interventions to facilitate the inclusion of students with disabilities. However, little is known about the potential that have adapted and Paralympic sports as content to raise awareness of students without disabilities, especially in inclusive practice context. On the other hand, teachers’ attitude and their training are presented as key in this context because it is who selects the content to work. We understand that PE have a unique opportunity in the curriculum to encourage active and effective participation of students with disabilities in class (especially at motor competence development, etc.), although this depends on factors related to the two agents. For these reasons that the PE, as actors and as content is displayed as a context for investigating the processes of inclusion of students with disabilities in education. This research aims to shed light on the questions that condition and limit this context, from a multidisciplinary perspective, with different methodologies on the three agents mentioned. The lack of accord in the literature regarding the characteristics and type of effective facilitators of awareness of students, and that is a new area as a research topic, has prompted us to work in this topic research. The first aim of this research was to design and implement a program of awareness towards disability based on adapted sports in the area of physical education for middle and high school students. Initially, a literature search was performed both nationally and internationally, in order to define the main features that brings the scientific literature in this area. On the one hand, we supported in the Paralympic School Day (IPC, 2004) educative program and on the other hand, in that review, we developed an initial approach to structure and phases. The project was presented to the Spanish Paralympic Committee and the Spanish Sports Federations for people with disabilities, in order to obtain institutional support in the form of guarantees and resources not only in economic terms but also as logistical support and dissemination. Thanks to the support of Fundación Sanitas, Liberty Seguros and Politechnical University of Madrid, we proceeded to design the program. For the development of teaching resources it was contacted experts in the field of Adapted Physical Activity and physical education and both the field of education (high school teachers and university professors) as the adapted sport national. In turn, it began to spread among the teachers of the schools in order to identify their interest in participating in the academic year (2012-2013) in the "Inclusive Sport in School" program. With the completion of the development of educational materials to schools he was visited to present the briefing where the program, as well as features and steps for its implementation are explained. The program is based on the Contact Theory (Allport, 1954) and based on adapted and Paralympic sports, raised with an inclusive approach, selecting strategies for promoting awareness and awareness to inclusion like information, contact and simulation of disability. At the meeting held at the Faculty of Sciences of Physical Activity and Sport (INEF-UPM) in February 2013, it was coordinated with the teachers implementing the program in each of the 13 schools with concrete actions such as adequacy of methodological approach in the annual planning of the teacher, the loan or the presentation of materials Paralympian among other issues. To achieve the objective 2 of this paper, to analyze the effect of the program on the various actions involved it, students without disabilities, PE teachers and students with disabilities, the date for management of the different methodological tools for before (pretest) and after the intervention (posttets). For students without disabilities (N= 1068), the effect of the intervention on the attitude towards inclusion was analyzed, using the quantitative questionnaire "Integrated Physical Education Attitude Children-Revised" (CAIPE-R; Block, 1995), after validation and adaptation to the Spanish context. The results showed significant positive changes in the attitude of the group with no structured contact with students with disabilities. This shows the beliefs towards the cooperative play was with peers with disabilities in PE classes also measured, using the questionnaire "Children's Beliefs Toward Cooperative Playing With Peers With Disabilities in Physical Education" (CBIPPD-MPE; Obrusnikova, Block, and Dillon, 2010). The development of a system of categories based on the Theory of Planned Behavior (Azjen, 1991) served as the basis for analysis of the beliefs of students without disabilities. After surgery, emerging behavioral beliefs remained, except in the case of the factors identified as barriers to inclusion. In the case of normative beliefs also they remained after surgery and regarding control beliefs, students identified the teacher as the main facilitator of inclusion. Regarding PE teachers participating in the program (N = 18), the effect of the program was analyzed their attitude toward inclusion of students with disability in PE with the questionnaire "Toward Attitude inclusion of Individual with in Physical Education "(ATISDPE-R; Kudláèek, Válková, Sherrill, Myers, and French, 2002). The results showed no significant difference occurred after surgery in the general attitude, finding some differences in certain related benefits of inclusion in students without disability relating to teachers with previous experience with disability in PE before intervention. The other dimension was analyzed the effect of the intervention on self-efficacy of teachers in the teaching of PE in inclusive terms, having used the questionnaire "Self-efficacy in PE teaching even under conditions" (SEIPE; Hutzler, Zach, and Gafni, 2005). The results showed significant differences positive in issues like being able to enhance the optimal conditions for teaching students with physical disabilities as amputation and severe visual impairment in both sports situations, such as games or activities outside the school to for teachers. Regard to gender, men earned higher values regarding women about feel more able to include students with physical disabilities in both games during recess and teaching sports technique. Teachers with less than 10 years of teaching showed more positive values as you feel able to include a student with physical disabilities in sports during their leisure time. The analysis of daily teacher shows on the one hand, emerging trends as key facilitators or barrier of the inclusion elements in PE, identifying the students without disabilities themselves, the professor, contents, materials and organization. Furthermore, the analysis of daily teacher about the contents proposed in the program. In the case of students with disabilities (N=22), the impact of the intervention program on self-concept was analyzed, with the questionnaire "Self-concept form 5" (AF5, F. Garcia and Musitu, 2001). The women showed significant differences before the intervention in family dimension, while men scored higher values in the social and physical dimensions were found. In terms of age, significant differences were found before the intervention, with higher values in younger students (12-14 years) in the physical dimension, while older children (15-17 years) showed higher values the social dimension of the questionnaire. Respect disabilities, students with motor disabilities showed better values than those with hearing impairment to the physical dimension before surgery. As for the general self-concept, positive significant differences occur in the academic dimension. As for the effect of the program on self-esteem of students with disabilities Scale "Rosenberg Self-Esteem" (Rosenberg, 1989) was used, not getting significant differences in gender. Only appear significant difference before the intervention in the case of younger students as they wish to be valued more, and students with hearing disabilities who do not feel very proud of themselves. Improved self-esteem generally occurs in that they feel less useless after surgery. With regard to the aim 3 of this research, after analyzing the results and have discussed them with the authors, it emerged the proposal of guidelines for both intervention program EF for sensitization and awareness of students towards inclusion as in the face of specific training for teachers, as key in such interventions. We believe that "Inclusive Sport Schools" program becomes a transforming element of reality, as it responds to the needs identified in the light of this research and come to respond to the various elements involved in its development. On the one hand, it meets the demand of the education sector in terms of the needs of teacher training, awareness of students without disability, and facilitates opportunities for students with disabilities for active participation in PE class. On the other hand, it meets the demand of sports institutions in the field of disability regarding the promotion and dissemination of adapted and Paralympic sports. Finally, it is shown as a resource from the university level for the training of degree in Physical Activity and Sport Science students, by its direct involvement with disability. For these reasons, this work is shown as a good starting point to further advance research in this area.

Three metal ions at the active site of the Tetrahymena group I ribozyme

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3′-oxygen of the oligonucleotide substrate and the 3′- and 2′-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezer

The non-Mendelian inheritance of organelle genes is a phenomenon common to almost all eukaryotes, and in the isogamous alga Chlamydomonas reinhardtii, chloroplast (cp) genes are transmitted from the mating type positive (mt+) parent. In this study, the preferential disappearance of the fluorescent cp nucleoids of the mating type negative (mt−) parent was observed in living young zygotes. To study the change in cpDNA molecules during the preferential disappearance, the cpDNA of mt+ or mt− origin was labeled separately with bacterial aadA gene sequences. Then, a single zygote with or without cp nucleoids was isolated under direct observation by using optical tweezers and investigated by nested PCR analysis of the aadA sequences. This demonstrated that cpDNA molecules are digested completely during the preferential disappearance of mt− cp nucleoids within 10 min, whereas mt+ cpDNA and mitochondrial DNA are protected from the digestion. These results indicate that the non-Mendelian transmission pattern of organelle genes is determined immediately after zygote formation.

Long-term potentiation activates the GAP-43 promoter: Selective participation of hippocampal mossy cells

Perforant path long-term potentiation (LTP) in intact mouse hippocampal dentate gyrus increased the neuron-specific, growth-associated protein GAP-43 mRNA in hilar cells 3 days after tetanus, but surprisingly not in granule cells, the perforant path target. This increase was positively correlated with level of enhancement and restricted to central hilar cells on the side of stimulation. Blockade of LTP by puffing dl-aminophosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor blocker into the molecular layer, eliminated LTP-induced GAP-43 mRNA elevation in hilar cells. To determine whether the mRNA elevation was mediated by transcription, LTP was studied in transgenic mice bearing a GAP-43 promoter-lacZ reporter gene. Promoter activity as indexed by Transgene expression (PATE) increased as indicated by blue staining of the lacZ gene product, β-galactosidase. Potentiation induced a blue band bilaterally in the inner molecular layer of the dentate gyrus along the entire septotemporal axis. Because mossy cells are the only neurons in the central hilar zone that project to the inner molecular layer bilaterally along the entire septotemporal axis and LTP-induced activation of PATE in this zone was confined to the side of stimulation, we concluded that mossy cells were unilaterally activated, increasing synthesis of β-galactosidase, which was transported bilaterally. Neither granule cells nor pyramidal cells demonstrated increased PATE or increased GAP-43 mRNA levels. These results and recent evidence indicating the necessity of hilar neurons for LTP point to previously unheralded mossy cells as potentially critical for perforant path LTP and the GAP-43 in these cells as important for LTP persistence lasting days.

Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events

Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events. In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of the yeast kinetochore. Although Bir1p carries two BIR motifs in the N-terminal region, the C-terminal third of the protein is sufficient to provide strong interaction with Ndc10p and moderate interaction with Skp1p, another essential component of the yeast kinetochore. In addition, deletion of BIR1 is synthetically lethal with deletion of CBF1 or CTF19, genes specifying two other components of the yeast kinetochore. Yeast cells deleted of BIR1 have a chromosome-loss phenotype, which can be completely rescued by elevating NDC10 dosage. Furthermore, overexpression of either full-length or the C-terminal region of Bir1p can efficiently suppress the chromosome-loss phenotype of both bir1Δ null and skp1-4 mutants. Our data suggest that Bir1p participates in chromosome segregation events, either directly or via interaction with kinetochore proteins, and these effects are apparently not mediated by the BIR domains of Bir1p.

Structural evidence for a programmed general base in the active site of a catalytic antibody

The crystal structure of the complex of a catalytic antibody with its cationic hapten at 1.9-Å resolution demonstrates that the hapten amidinium group is stabilized through an ionic pair interaction with the carboxylate of a combining-site residue. The location of this carboxylate allows it to act as a general base in an allylic rearrangement. When compared with structures of other antibody complexes in which the positive moiety of the hapten is stabilized mostly by cation–π interactions, this structure shows that the amidinium moiety is a useful candidate to elicit a carboxylate in an antibody combining site at a predetermined location with respect to the hapten. More generally, this structure highlights the advantage of a bidentate hapten for the programmed positioning of a chemically reactive residue in an antibody through charge complementarity to the hapten.

Participation of Mitochondrial Metabolism in Photorespiration1 : Reconstituted System of Peroxisomes and Mitochondria from Spinach Leaves

In this study the interplay of mitochondria and peroxisomes in photorespiration was simulated in a reconstituted system of isolated mitochondria and peroxisomes from spinach (Spinacia oleracea L.) leaves. The mitochondria oxidizing glycine produced serine, which was reduced in the peroxisomes to glycerate. The required reducing equivalents were provided by the mitochondria via the malate-oxaloacetate (OAA) shuttle, in which OAA was reduced in the mitochondrial matrix by NADH generated during glycine oxidation. The rate of peroxisomal glycerate formation, as compared with peroxisomal protein, resembled the corresponding rate required during leaf photosynthesis under ambient conditions. When the reconstituted system produced glycerate at this rate, the malate-to-OAA ratio was in equilibrium with a ratio of NADH/NAD of 8.8 × 10−3. This low ratio is in the same range as the ratio of NADH/NAD in the cytosol of mesophyll cells of intact illuminated spinach leaves, as we had estimated earlier. This result demonstrates that in the photorespiratory cycle a transfer of redox equivalents from the mitochondria to peroxisomes, as postulated from separate experiments with isolated mitochondria and peroxisomes, can indeed operate under conditions of the very low reductive state of the NADH/NAD system prevailing in the cytosol of mesophyll cells in a leaf during photosynthesis.

Functional dynamics in the active site of the ribonuclease binase

Binase, a member of a family of microbial guanyl-specific ribonucleases, catalyzes the endonucleotic cleavage of single-stranded RNA. It shares 82% amino acid identity with the well-studied protein barnase. We used NMR spectroscopy to study the millisecond dynamics of this small enzyme, using several methods including the measurement of residual dipolar couplings in solution. Our data show that the active site of binase is flanked by loops that are flexible at the 300-μs time scale. One of the catalytic residues, His-101, is located on such a flexible loop. In contrast, the other catalytic residue, Glu-72, is located on a β-sheet, and is static. The residues Phe-55, part of the guanine base recognition site, and Tyr-102, stabilizing the base, are the most dynamic. Our findings suggest that binase possesses an active site that has a well-defined bottom, but which has sides that are flexible to facilitate substrate access/egress, and to deliver one of the catalytic residues. The motion in these loops does not change on complexation with the inhibitor d(CGAG) and compares well with the maximum kcat (1,500 s−1) of these ribonucleases. This observation indicates that the NMR-measured loop motions reflect the opening necessary for product release, which is apparently rate limiting for the overall turnover.

Participation of recombination proteins in rescue of arrested replication forks in UV-irradiated Escherichia coli need not involve recombination

Alternative reproductive cycles make use of different strategies to generate different reproductive products. In Escherichia coli, recA and several other rec genes are required for the generation of recombinant genomes during Hfr conjugation. During normal asexual reproduction, many of these same genes are needed to generate clonal products from UV-irradiated cells. However, unlike conjugation, this latter process also requires the function of the nucleotide excision repair genes. Following UV irradiation, the recovery of DNA replication requires uvrA and uvrC, as well as recA, recF, and recR. The rec genes appear to be required to protect and maintain replication forks that are arrested at DNA lesions, based on the extensive degradation of the nascent DNA that occurs in their absence. The products of the recJ and recQ genes process the blocked replication forks before the resumption of replication and may affect the fidelity of the recovery process. We discuss a model in which several rec gene products process replication forks arrested by DNA damage to facilitate the repair of the blocking DNA lesions by nucleotide excision repair, thereby allowing processive replication to resume with no need for strand exchanges or recombination. The poor survival of cellular populations that depend on recombinational pathways (compared with that in their excision repair proficient counterparts) suggests that at least some of the rec genes may be designed to function together with nucleotide excision repair in a common and predominant pathway by which cells faithfully recover replication and survive following UV-induced DNA damage.

Evidence that replication fork components catalyze establishment of cohesion between sister chromatids

Accurate chromosome segregation requires that replicated sister chromatids are held together until anaphase, when their “cohesion” is dissolved, and they are pulled to opposite spindle poles by microtubules. Establishment of new cohesion between sister chromatids in the next cell cycle is coincident with replication fork passage. Emerging evidence suggests that this temporal coupling is not just a coincident timing of independent events, but rather that the establishment of cohesion is likely to involve the active participation of replication-related activities. These include PCNA, a processivity clamp for some DNA polymerases, Trf4/Pol σ (formerly Trf4/Polκ), a novel and essential DNA polymerase, and a modified Replication Factor C clamp–loader complex. Here we describe recent advances in how cohesion establishment is linked to replication, highlight important unanswered questions in this new field, and describe a “polymerase switch” model for how cohesion establishment is coupled to replication fork progression. Building the bridges between newly synthesized sister chromatids appears to be a fundamental but previously unrecognized function of the eukaryotic replication machinery.

The entry and clearance of Ca2+ at individual presynaptic active zones of hair cells from the bullfrog's sacculus.

Neurotransmitter is released when Ca2+ triggers the fusion of synaptic vesicles with the plasmalemma. To study factors that regulate Ca2+ concentration at the presynaptic active zones of hair cells, we used laser-scanning confocal microscopy with the fluorescent Ca2+ indicator fluo 3. The experimental results were compared with the predictions of a model of presynaptic Ca2+ concentration in which Ca2+ enters a cell through a point source, diffuses from the entry site, and binds to fixed or mobile Ca2+ buffers. The observed time course and magnitude of fluorescence changes under a variety of conditions were well fit when the model included mobile molecules as the only Ca2+ buffer. The results confirm the localized entry of Ca2+ underlying neurotransmitter release and suggest that Ca2+ is cleared from an active zone almost exclusively by mobile buffer.

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pKa values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.