The Zero-Removing Property and Lagrange-Type Interpolation Series

The classical Kramer sampling theorem, which provides a method for obtaining orthogonal sampling formulas, can be formulated in a more general nonorthogonal setting. In this setting, a challenging problem is to characterize the situations when the obtained nonorthogonal sampling formulas can be expressed as Lagrange-type interpolation series. In this article a necessary and sufficient condition is given in terms of the zero removing property. Roughly speaking, this property concerns the stability of the sampled functions on removing a finite number of their zeros.

Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.

La transformación de las grandes estaciones europeas con la llegada de la Alta Velocidad. El caso de Atocha

La aparición del tren de alta velocidad en Europa en las últimas décadas del siglo XX supuso el resurgir de un medio de transporte en progresivo declive desde la popularización del automóvil y del avión. La decadencia del ferrocarril había supuesto en muchos casos el abandono, o incluso la demolición, de estaciones históricas y el deterioro de su entorno urbano. Como reacción a esa desatención surgió, también en el último cuarto de siglo, una mayor conciencia social preocupada por la conservación del patrimonio construido del ferrocarril. La necesidad de adaptación de las grandes estaciones de ferrocarril para dar servicio al nuevo sistema de transporte, junto con el interés por poner en valor sus construcciones históricas y su céntrico entorno, ha dado como resultado la realización de importantes transformaciones. El objeto de la presente investigación es el estudio de las transformaciones que han sufrido las grandes estaciones europeas del siglo XIX con la llegada del tren de alta velocidad, profundizando de manera especial en el caso más significativo que tenemos en nuestro país: la estación de Atocha. En el ámbito europeo es donde se localizan los ejemplos más relevantes de estaciones que tuvieron gran trascendencia en el siglo XIX y que ahora, con la llegada de la Alta Velocidad, vuelven a recuperar su grandeza. En España, el crecimiento de la Alta Velocidad en los últimos años ha sido extraordinario, hasta situarse como el segundo país del mundo con más kilómetros de líneas de alta velocidad en operación y, en consecuencia, se ha construido un gran número de estaciones adaptadas a este servicio. El caso más notable es el de la estación de Atocha, que desde la llegada del AVE en 1992 hasta el día de hoy, se ha convertido en uno de los complejos ferroviarios más importantes del mundo. El trabajo parte del estudio de otros referentes europeos, como las Gares de París, la estación de St Pancras en Londres y de otras cinco estaciones del centro de Europa –Amsterdam Centraal, Antwerpen Centraal, Köln Hauptbahnhof, Frankfurt (Main) Hauptbahnhof y la Gare de Strasbourg–, para establecer el marco analítico sobre el que se profundiza con la estación de Atocha. El proceso de transformación de la estación de Atocha se ha gestado a través de una serie de proyectos que han ido configurando la estación hasta el momento actual y planteando la previsión de futuro: el proyecto del Plan General de Madrid, el concurso de ideas para el diseño de la estación, la estación de Cercanías, la estación de Alta Velocidad y Largo Recorrido, la ampliación de esta para separar los flujos por niveles, los Estudios Informativos del Nuevo Complejo Ferroviario de la Estación de Atocha y su primera fase de construcción. Estos siete proyectos son objeto de un análisis en tres niveles: análisis cronológico, análisis funcional y análisis formal. La estación de Atocha fue la primera estación histórica europea en sufrir una gran transformación vinculada a la llegada de la Alta Velocidad. Aporta el entendimiento de la estación como un todo y la intermodalidad como sus principales valores, además de la gran mejora urbana que supuso la «operación Atocha», y adolece de ciertas carencias en su desarrollo comercial, vinculadas en parte a la presencia del jardín tropical, y de un pobre espacio en las salas de embarque para los pasajeros de salidas. La estación de Atocha completa su transformación a partir de su renovación funcional, manteniendo la carga simbólica de su historia. De la confrontación del caso de Atocha con otras importantes estaciones europeas resulta la definición de las principales consecuencias de la llegada de la Alta Velocidad a las grandes terminales europeas y la identificación de los elementos clave en su transformación. Las consecuencias principales son: la potenciación de la intermodalidad con otros medios de transporte, el desarrollo comercial no necesariamente destinado a los usuarios de los servicios ferroviarios, y la puesta en valor de la antigua estación y de su entorno urbano. Por su parte, los elementos clave en la transformación de las grandes estaciones tienen que ver directamente con la separación de flujos, el entendimiento de la estación por niveles, la dotación de nuevos accesos laterales y la construcción de una nueva gran cubierta para los nuevos andenes. La preeminencia de unos elementos sobre otros depende del carácter propio de cada estación y de cada país, de la magnitud de la intervención y, también, de la estructura y composición de los equipos encargados del diseño de la nueva estación. En la actualidad, nos encontramos en un momento interesante respecto a las estaciones de Alta Velocidad. Tras el reciente atentado frustrado en el Thalys que viajaba de Ámsterdam a París, se ha acordado establecer controles de identidad y equipajes en todas las estaciones de la red europea de alta velocidad, lo que implicará modificaciones importantes en las grandes estaciones que, probablemente, tomarán el modelo de la estación de Atocha como referencia. ABSTRACT The emergence of the high speed train in Europe in the last few decades of the 20th century represented the resurgence of a means of transport in progressive decline since the popularization of the car and the airplane. The railway decay brought in many cases the abandonment, or even the demolition, of historical stations and the deterioration of its urban environment. In response to that neglect, a greater social awareness towards the preservation of the railway built heritage raised up, also in the last quarter-century. The need for adaptation of the great railway stations to serve the new transport system, along with the interest in enhancing the historical buildings and its central locations, had resulted in important transformations. The subject of current investigation is the study of the transformations that the great 19th century European stations have experienced with the arrival of the high speed rail, deepening in particular in the most significant case we have in Spain: Atocha railway station. At European level is where the most relevant examples of stations which have had a great significance in the 19th century and now, with the arrival of the high speed train, have regain their greatness, are located. In Spain, the growth of the high speed rail over the past few years has been outstanding. Today is the second country in the world with the longest high speed rail network in operation and, therefore, with a great number of new stations adapted to this service. The most remarkable case is Atocha station. Since the arrival of the AVE in 1992, the station has become one of the world's most important railway hub. The research starts with the study of other European reference points, as the Gares of Paris, St Pancras station in London and five other stations of Central Europe –Amsterdam Centraal, Antwerpen Centraal, Köln Hauptbahnhof, Frankfurt (Main) Hauptbahnhof y la Gare de Strasbourg–, to establish the analytical framework that will be deepen with Atocha station. The transformation process of Atocha station has been created through a number of projects that have forged the station to date and have raised the sights in the future: the project of the General Urban Development Plan, the ideas competition for the station design, the Suburban train station, the High Speed and Long Distance station, its enlargement in order to separate passenger flows in different levels, the 'Masterplans' for the new Atocha transport hub and its first phase of construction. These seven projects are under scrutiny at three levels: chronological analysis, functional analysis and formal analysis. Atocha station was the first European historical station to undergo a great transformation tied to the arrival of the high speed rail. It brings the understanding of the station as a whole and the intermodality as its greatest values, besides the great urban improvement of the 'Atocha operation', and suffers from certain shortcomings in its commercial development, partly linked to the presence of the tropical garden, and from a poor space in the departure lounges. Atocha station completes its transformation on the basis of its functional renewal, keeping the symbolic charge of its history. The confrontation of Atocha case with the great European stations results in the definition of the principal consequences of the high speed rail arrival to the great European terminals and the identification of the key elements in its transformation. The principal consequences are: the empowering of the intermodality with other means of transport, of the commercial development, not necessarily intended for railway services users, and the enhancement of the old station and its urban environment. On the other hand, the key elements in the transformation of the great stations are directly related with the separation of passenger flows, the understanding of the station in different levels, the placement of new lateral accesses and the construction of a new deck over the new platforms. The pre-eminence of some elements over the others depends on the particular nature of each station and each country, on the scale of the intervention and also in the structure and composition of the teams in charge of the new station design. Nowadays, this is an interesting time concerning the high speed rail stations. After the recent foiled terrorist attempt in the Thalys train travelling from Amsterdam to Paris, it was agreed to establish passenger and luggage controls in every European high speed rail station. This will mean important changes in these great stations, which probably will take Atocha station's model as a reference.

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein

Protein–protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the β-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 μM IC50. Structural analysis by NMR showed that both the backbone of the chimeric β-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC50 of 0.1–1.0 μM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4+ cells by different virus isolates. Thus, core regions of large protein–protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein–protein interactions that may represent useful tools in biology and in drug discovery.

Activity-based protein profiling: The serine hydrolases

With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.

Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive “glycomodules” extensively O-glycosylated through the Hyp residues. The patterns of Hyp-O-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1–5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test, we designed two simple HRGPs as fusion proteins with green fluorescent protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.

Wild-type Escherichia coli grows on the chitin disaccharide, N,N′-diacetylchitobiose, by expressing the cel operon

We report here that wild-type Escherichia coli can grow on the chitin disaccharide, N,N′-diacetylchitobiose (GlcNAc)2, as the sole source of carbon. Transposon mutants were isolated that were unable to ferment (GlcNAc)2 but grew normally on the monosaccharide GlcNAc. One such mutant was used to screen a wild-type E. coli genomic cosmid library for restoration of (GlcNAc)2 fermentation. A partial sequence analysis of the isolated fragment mapped the clone to the (previously sequenced) E. coli genome between 39.0 and 39.2 min. The nucleotide ORFs at this region had been previously assigned to code for a “cryptic” cellobiose utilization (cel) operon. We report here, however, that functional analysis of the operon, including growth and chemotaxis, reveal that it encodes a set of proteins that are not cryptic, but are induced by (GlcNAc)2 and catabolize the disaccharide. We therefore propose to rename the cel operon as the chb (N,N′-diacetylchitobiose) operon, with the letter designation of the genes of the operon to be reassigned consistent with the nomenclature based on functional characterization of the gene products as follows: celA to chbB, celB to chbC, celC to chbA, celD to chbR, and celF to chbF. Furthermore, sequencing evidence indicates that the operon contains an additional gene of unknown function to be designated as chbG. Thus, the overall gene sequence is to be named chbBCARFG.

Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

We report here the isolation and functional analysis of the rfc3+ gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3+ gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3+ is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

Rapid generation of nested chromosomal deletions on mouse chromosome 2

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F1 hybrid embryonic stem cell line, F1(129S1×Cast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.

Genomic organization of α1 and β1 subunits of the mammalian soluble guanylyl cyclase genes

The structures of the genes encoding the α1 and β1 subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNAs isolated from mouse lungs encoding the α1 (2.5 kb) and β1 (3.3 kb) subunits are presented in this report. The α1 sGC gene is approximately 26.4 kb and contains nine exons, whereas the β1 sGC gene spans 22 kb and consists of 14 exons. The positions of exon/intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome Database predicted the exon/intron boundaries of the human genes and revealed that human and mouse α1 and β1 sGC genes have similar structures. Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5′ untranscribed regions of α1 and β1 subunit genes were subcloned into luciferase reporter constructs, and the functional analysis of promoter activity was performed in murine neuroblastoma N1E-115 cells. Our results indicate that the 5′ untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, suggest independent regulation of both genes.

Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.

The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492–742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds1

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3β-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3β-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3β-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.

cAMP-response element modulator tau is a positive regulator of testis angiotensin converting enzyme transcription.

Testis angiotensin-converting enzyme (ACE) is a unique form of ACE, only produced by male germ cells, and results from a testis-specific promoter found within the ACE gene. We have investigated the role of cAMP-response element modulator (CREM)tau in testis ACE transcription. In gel shift experiments, testes nuclear proteins retard an oligonucleotide containing the cAMP-response element (CRE) found at position -55 in the testis ACE promoter. Anti-CREM antibody supershifts this complex. Competitive gel shift shows that recombinant CREM tau protein and testis nuclear proteins have a similar specificity of binding to the tests ACE CRE. Functional analysis using in vitro transcription and transfection studies also demonstrate that CREM tau protein is a transcriptional activator of the testis ACE promoter. Western blot analysis identifies CREM tau protein in the protein-DNA complex formed between nuclear proteins and the testis ACE CRE motif. This analysis also identified other CREM isoforms in the gel-shifted complex, which are thought to be CREM tau 1/2, CREM alpha/beta, and S-CREM. These data indicate that CREM tau isoforms play an important role as a positive regulator in the tissue-specific expression of testis ACE.