Experimental results on multicarrier SIMO HF communications

Achieving reliable communication over HF channels is known to be challenging due to the particularly hostile propagation medium. To address this problem, diversity techniques were shown to be promising. In this paper, we demonstrate through experimental results the benefits of different diversity strategies when applied to multi-input-multi-output (MIMO) multicarrier systems. The performance gains of polarisation, space and frequency diversities are quantified using different measurement campaigns.

An algorithm for graphical computer results in shells

A Finite Element technique to interpolate general data (function values and its derivatives) has been developped. The technique can be considered as a generalized solution of the classical polynomial interpolation, because the condition for the interpolating function to be a polynomial is replaced by a minimizing condition of a given “smoothing” functional. In this way it is possible to find interpolating functions with a given level of continuity according to the class of finite elements used. Examples have been presented in order to assess the accuracy and efficiency of the procedure.

Metodología de análisis estadístico de roturas en redes de distribución de agua

La presente Tesis plantea una metodología de análisis estadístico de roturas de tubería en redes de distribución de agua, que analiza la relación entre las roturas y la presión de agua y que propone la implantación de una gestión de presiones que reduzca el número de roturas que se producen en dichas redes. Las redes de distribución de agua se deterioran y una de sus graves consecuencias es la aparición de roturas frecuentes en sus tuberías. Las roturas llevan asociados elevados costes sociales, económicos y medioambientales y es por ello por lo que las compañías gestoras del agua tratan de reducirlas en la medida de lo posible. Las redes de distribución de agua se pueden dividir en zonas o sectores que facilitan su control y que pueden ser independientes o aislarse mediante válvulas, como ocurre en las redes de países más desarrollados, o pueden estar intercomunicados hidráulicamente. La implantación de una gestión de presiones suele llevarse a cabo a través de las válvulas reductoras de presión (VPR), que se instalan en las cabeceras de estos sectores y que controlan la presión aguas abajo de la misma, aunque varíe su caudal de entrada. Los métodos más conocidos de la gestión de presiones son la reducción de presiones, que es el control más habitual, el mantenimiento de la presión, la prevención y/o alivio de los aumentos repentinos de presión y el establecimiento de un control por alturas. A partir del año 2005 se empezó a reconocer el efecto de la gestión de presiones sobre la disminución de las roturas. En esta Tesis, se sugiere una gestión de presiones que controle los rangos de los indicadores de la presión de cabecera que más influyan en la probabilidad de roturas de tubería. Así, la presión del agua se caracteriza a través de indicadores obtenidos de la presión registrada en la cabecera de los sectores, debido a que se asume que esta presión es representativa de la presión de operación de todas las tuberías porque las pérdidas de carga son relativamente bajas y las diferencias topográficas se tienen en cuenta en el diseño de los sectores. Y los indicadores de presión, que se pueden definir como el estadístico calculado a partir de las series de la presión de cabecera sobre una ventana de tiempo, pueden proveer la información necesaria para ayudar a la toma de decisiones a los gestores del agua con el fin de reducir las roturas de tubería en las redes de distribución de agua. La primera parte de la metodología que se propone en esta Tesis trata de encontrar los indicadores de presión que influyen más en la probabilidad de roturas de tuberías. Para conocer si un indicador es influyente en la probabilidad de las roturas se comparan las estimaciones de las funciones de distribución acumulada (FDAs) de los indicadores de presiones, considerando dos situaciones: cuando se condicionan a la ocurrencia de una rotura (suceso raro) y cuando se calculan en la situación normal de operación (normal operación). Por lo general, las compañías gestoras cuentan con registros de roturas de los años más recientes y al encontrarse las tuberías enterradas se complica el acceso a la información. Por ello, se propone el uso de funciones de probabilidad que permiten reducir la incertidumbre asociada a los datos registrados. De esta forma, se determinan las funciones de distribución acumuladas (FDAs) de los valores del indicador de la serie de presión (situación normal de operación) y las FDAs de los valores del indicador en el momento de ocurrencia de las roturas (condicionado a las roturas). Si las funciones de distribución provienen de la misma población, no se puede deducir que el indicador claramente influya en la probabilidad de roturas. Sin embargo, si se prueba estadísticamente que las funciones proceden de la misma población, se puede concluir que existe una relación entre el indicador analizado y la ocurrencia de las roturas. Debido a que el número de valores del indicador de la FDA condicionada a las roturas es mucho menor que el número de valores del indicador de la FDA incondicional a las roturas, se generan series aleatorias a partir de los valores de los indicadores con el mismo número de valores que roturas registradas hay. De esta forma, se comparan las FDAs de series aleatorias del indicador con la FDA condicionada a las roturas del mismo indicador y se deduce si el indicador es influyente en la probabilidad de las roturas. Los indicadores de presión pueden depender de unos parámetros. A través de un análisis de sensibilidad y aplicando un test estadístico robusto se determina la situación en la que estos parámetros dan lugar a que el indicador sea más influyente en la probabilidad de las roturas. Al mismo tiempo, los indicadores se pueden calcular en función de dos parámetros de cálculo que se denominan el tiempo de anticipación y el ancho de ventana. El tiempo de anticipación es el tiempo (en horas) entre el final del periodo de computación del indicador de presión y la rotura, y el ancho de ventana es el número de valores de presión que se requieren para calcular el indicador de presión y que es múltiplo de 24 horas debido al comportamiento cíclico diario de la presión. Un análisis de sensibilidad de los parámetros de cálculo explica cuándo los indicadores de presión influyen más en la probabilidad de roturas. En la segunda parte de la metodología se presenta un modelo de diagnóstico bayesiano. Este tipo de modelo forma parte de los modelos estadísticos de prevención de roturas, parten de los datos registrados para establecer patrones de fallo y utilizan el teorema de Bayes para determinar la probabilidad de fallo cuando se condiciona la red a unas determinadas características. Así, a través del teorema de Bayes se comparan la FDA genérica del indicador con la FDA condicionada a las roturas y se determina cuándo la probabilidad de roturas aumenta para ciertos rangos del indicador que se ha inferido como influyente en las roturas. Se determina un ratio de probabilidad (RP) que cuando es superior a la unidad permite distinguir cuándo la probabilidad de roturas incrementa para determinados intervalos del indicador. La primera parte de la metodología se aplica a la red de distribución de la Comunidad de Madrid (España) y a la red de distribución de Ciudad de Panamá (Panamá). Tras el filtrado de datos se deduce que se puede aplicar la metodología en 15 sectores en la Comunidad de Madrid y en dos sectores, llamados corregimientos, en Ciudad de Panamá. Los resultados demuestran que en las dos redes los indicadores más influyentes en la probabilidad de las roturas son el rango de la presión, que supone la diferencia entre la presión máxima y la presión mínima, y la variabilidad de la presión, que considera la propiedad estadística de la desviación típica. Se trata, por tanto, de indicadores que hacen referencia a la dispersión de los datos, a la persistencia de la variación de la presión y que se puede asimilar en resistencia de materiales a la fatiga. La segunda parte de la metodología se ha aplicado a los indicadores influyentes en la probabilidad de las roturas de la Comunidad de Madrid y se ha deducido que la probabilidad de roturas aumenta para valores extremos del indicador del rango de la presión y del indicador de la variabilidad de la presión. Finalmente, se recomienda una gestión de presiones que limite los intervalos de los indicadores influyentes en la probabilidad de roturas que incrementen dicha probabilidad. La metodología propuesta puede aplicarse a otras redes de distribución y puede ayudar a las compañías gestoras a reducir el número de fallos en el sistema a través de la gestión de presiones. This Thesis presents a methodology for the statistical analysis of pipe breaks in water distribution networks. The methodology studies the relationship between pipe breaks and water pressure, and proposes a pressure management procedure to reduce the number of breaks that occur in such networks. One of the manifestations of the deterioration of water supply systems is frequent pipe breaks. System failures are one of the major challenges faced by water utilities, due to their associated social, economic and environmental costs. For all these reasons, water utilities aim at reducing the problem of break occurrence to as great an extent as possible. Water distribution networks can be divided into areas or sectors, which facilitates the control of the network. These areas may be independent or isolated by valves, as it usually happens in developing countries. Alternatively, they can be hydraulically interconnected. The implementation of pressure management strategies is usually carried out through pressure-reducing valves (PRV). These valves are installed at the head of the sectors and, although the inflow may vary significantly, they control the downstream pressure. The most popular methods of pressure management consist of pressure reduction, which is the common form of control, pressure sustaining, prevention and/or alleviation of pressure surges or large variations in pressure, and level/altitude control. From 2005 onwards, the effects of pressure management on burst frequencies have become more widely recognized in the technical literature. This thesis suggests a pressure management that controls the pressure indicator ranges most influential on the probability of pipe breaks. Operating pressure in a sector is characterized by means of a pressure indicator at the head of the DMA, as head losses are relatively small and topographical differences were accounted for at the design stage. The pressure indicator, which may be defined as the calculated statistic from the time series of pressure head over a specific time window, may provide necessary information to help water utilities to make decisions to reduce pipe breaks in water distribution networks. The first part of the methodology presented in this Thesis provides the pressure indicators which have the greatest impact on the probability of pipe breaks to be determined. In order to know whether a pressure indicator influences the probability of pipe breaks, the proposed methodology compares estimates of cumulative distribution functions (CDFs) of a pressure indicator through consideration of two situations: when they are conditioned to the occurrence of a pipe break (a rare event), and when they are not (a normal operation). Water utilities usually have a history of failures limited to recent periods of time, and it is difficult to have access to precise information in an underground network. Therefore, the use of distribution functions to address such imprecision of recorded data is proposed. Cumulative distribution functions (CDFs) derived from the time series of pressure indicators (normal operation) and CDFs of indicator values at times coincident with a reported pipe break (conditioned to breaks) are compared. If all estimated CDFs are drawn from the same population, there is no reason to infer that the studied indicator clearly influences the probability of the rare event. However, when it is statistically proven that the estimated CDFs do not come from the same population, the analysed indicator may have an influence on the occurrence of pipe breaks. Due to the fact that the number of indicator values used to estimate the CDF conditioned to breaks is much lower in comparison with the number of indicator values to estimate the CDF of the unconditional pressure series, and that the obtained results depend on the size of the compared samples, CDFs from random sets of the same size sampled from the unconditional indicator values are estimated. Therefore, the comparison between the estimated CDFs of random sets of the indicator and the estimated CDF conditioned to breaks allows knowledge of if the indicator is influential on the probability of pipe breaks. Pressure indicators depend on various parameters. Sensitivity analysis and a robust statistical test allow determining the indicator for which these parameters result most influential on the probability of pipe breaks. At the same time, indicators can be calculated according to two model parameters, named as the anticipation time and the window width. The anticipation time refers to the time (hours) between the end of the period for the computation of the pressure indicator and the break. The window width is the number of instantaneous pressure values required to calculate the pressure indicator and is multiple of 24 hours, as water pressure has a cyclical behaviour which lasts one day. A sensitivity analysis of the model parameters explains when the pressure indicator is more influential on the probability of pipe breaks. The second part of the methodology presents a Bayesian diagnostic model. This kind of model belongs to the class of statistical predictive models, which are based on historical data, represent break behavior and patterns in water mains, and use the Bayes’ theorem to condition the probability of failure to specific system characteristics. The Bayes’ theorem allows comparing the break-conditioned FDA and the unconditional FDA of the indicators and determining when the probability of pipe breaks increases for certain pressure indicator ranges. A defined probability ratio provides a measure to establish whether the probability of breaks increases for certain ranges of the pressure indicator. The first part of the methodology is applied to the water distribution network of Madrid (Spain) and to the water distribution network of Panama City (Panama). The data filtering method suggests that the methodology can be applied to 15 sectors in Madrid and to two areas in Panama City. The results show that, in both systems, the most influential indicators on the probability of pipe breaks are the pressure range, which is the difference between the maximum pressure and the minimum pressure, and pressure variability, referred to the statistical property of the standard deviation. Therefore, they represent the dispersion of the data, the persistence of the variation in pressure and may be related to the fatigue in material resistance. The second part of the methodology has been applied to the influential indicators on the probability of pipe breaks in the water distribution network of Madrid. The main conclusion is that the probability of pipe breaks increases for the extreme values of the pressure range indicator and of the pressure variability indicator. Finally, a pressure management which limits the ranges of the pressure indicators influential on the probability of pipe breaks that increase such probability is recommended. The methodology presented here is general, may be applied to other water distribution networks, and could help water utilities reduce the number of system failures through pressure management.

Interval-based ranking in noisy evolutionary multiobjective optimization

As one of the most competitive approaches to multi-objective optimization, evolutionary algorithms have been shown to obtain very good results for many realworld multi-objective problems. One of the issues that can affect the performance of these algorithms is the uncertainty in the quality of the solutions which is usually represented with the noise in the objective values. Therefore, handling noisy objectives in evolutionary multi-objective optimization algorithms becomes very important and is gaining more attention in recent years. In this paper we present ?-degree Pareto dominance relation for ordering the solutions in multi-objective optimization when the values of the objective functions are given as intervals. Based on this dominance relation, we propose an adaptation of the non-dominated sorting algorithm for ranking the solutions. This ranking method is then used in a standardmulti-objective evolutionary algorithm and a recently proposed novel multi-objective estimation of distribution algorithm based on joint variable-objective probabilistic modeling, and applied to a set of multi-objective problems with different levels of independent noise. The experimental results show that the use of the proposed method for solution ranking allows to approximate Pareto sets which are considerably better than those obtained when using the dominance probability-based ranking method, which is one of the main methods for noise handling in multi-objective optimization.

Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two types of subunits: RNR1 contains the active site for reduction and the binding sites for the nucleotide allosteric effectors. RNR2 contains the diiron-tyrosyl radical (Y⋅) cofactor essential for the reduction process. Studies in yeast have recently identified four RNR subunits: Y1 and Y3, Y2 and Y4. These proteins have been expressed in Saccharomyces cerevisiae and in Escherichia coli and purified to ≈90% homogeneity. The specific activity of Y1 isolated from yeast and E. coli is 0.03 μmol⋅min−1⋅mg−1 and of (His)6-Y2 [(His)6-Y2-K387N] from yeast is 0.037 μmol⋅min−1⋅mg−1 (0.125 μmol⋅min−1⋅mg−1). Y2, Y3, and Y4 isolated from E. coli have no measurable activity. Efforts to generate Y⋅ in Y2 or Y4 using Fe2+, O2, and reductant have been unsuccessful. However, preliminary studies show that incubation of Y4 and Fe2+ with inactive E. coli Y2 followed by addition of O2 generates Y2 with a specific activity of 0.069 μmol⋅min−1⋅mg−1 and a Y⋅. A similar experiment with (His)6-Y2-K387N, Y4, O2, and Fe2+ results in an increase in its specific activity to 0.30 μmol⋅min−1⋅mg−1. Studies with antibodies to Y4 and Y2 reveal that they can form a complex in vivo. Y4 appears to play an important role in diiron-Y⋅ assembly of Y2.

A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zα has allowed identification of motifs common to this class of nucleic acid binding domain.

Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF2α—F2-iPs—in urine has reflected lipid peroxidation in humans. However, up to 64 F2-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F2-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F2-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF2α-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F2-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F2-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF2α-VI in hypercholesterolemia and of 8,12-iso-iPF2α-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.

Ligation of intestinal epithelial CD1d induces bioactive IL-10: Critical role of the cytoplasmic tail in autocrine signaling

The intestinal epithelium is anatomically positioned to serve as the critical interface between the lumen and the mucosal immune system. In addition to MHC class I and II antigens, intestinal epithelia constitutively express the nonclassical MHC molecule CD1d, a transmembrane molecule with a short cytoplasmic tail expressed as a β2-microglobulin-associated 48-kDa glycoprotein and novel β2-microglobulin-independent 37-kDa nonglycosylated protein on intestinal epithelia. At present, it is not known whether extracellular ligands can signal intestinal epithelial CD1d. To define signaling of CD1d cytoplasmic tail, retrovirus-mediated gene transfer was used to generate stable cell lines expressing wild-type CD1d or a chimeric molecule (extracellular CD1d and cytoplasmic CD1a), and surface CD1d was triggered by antibody crosslinking. Although wild-type CD1d was readily activated (tyrosine phosphorylation), no demonstrable signal was evident in cell lines expressing the chimeric molecule. Subsequent studies revealed that anti-CD1d crosslinking specifically induces epithelial IL-10 mRNA and protein and is blocked by the tyrosine kinase inhibitor genistein. Further studies addressing epithelial-derived IL-10 revealed that anti-CD1d crosslinking attenuates IFN-γ signaling and that such attenuation is reversed by addition of functionally inhibitory IL-10 antibodies. These results define signaling through surface CD1d, and, importantly, they demonstrate that this pathway may serve to dampen epithelial proinflammatory signals.

Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus

The α C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from the bca gene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

Conservation of the Drosophila lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi

Lysyl-tRNAs are essential for protein biosynthesis by ribosomal mRNA translation in all organisms. They are synthesized by lysyl-tRNA synthetases (EC 6.1.1.6), a group of enzymes composed of two unrelated families. In bacteria and eukarya, all known lysyl-tRNA synthetases are subclass IIc-type aminoacyl-tRNA synthetases, whereas some archaea have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. Examination of the preliminary genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetases. In contrast, no coding region with significant similarity to any class II-type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase-encoding gene is highly expressed, confirming that B. burgdorferi contains a functional class I-type lysyl-tRNA synthetase. The detection of an archaeal-type lysyl-tRNA synthetase in B. burgdorferi and other pathogenic spirochetes, but not to date elsewhere in bacteria or eukarya, indicates that the gene that encodes this enzyme has a common origin with its orthologue from the archaeal kingdom. This difference between the lysyl-tRNA synthetases of spirochetes and their hosts may be readily exploitable for the development of anti-spirochete therapeutics.

The Cell Surface Flocculin Flo11 Is Required for Pseudohyphae Formation and Invasion by Saccharomyces cerevisiae

Diploid yeast develop pseudohyphae in response to nitrogen starvation, while haploid yeast produce invasive filaments which penetrate the agar in rich medium. We have identified a gene, FLO11, that encodes a cell wall protein which is critically required for both invasion and pseudohyphae formation in response to nitrogen starvation. FLO11 encodes a cell surface flocculin with a structure similar to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. Cells of the Saccharomyces cerevisiae strain Σ1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids. In rich media, FLO11 is regulated by mating type; it is expressed in haploid cells but not in diploids. Upon transfer to nitrogen starvation media, however, FLO11 transcripts accumulate in diploid cells, but not in haploids. Overexpression of FLO11 in diploid cells, which are otherwise not invasive, enables them to invade agar. Thus, the mating type repression of FLO11 in diploids grown in rich media suffices to explain the inability of these cells to invade. The promoter of FLO11 contains a consensus binding sequence for Ste12p and Tec1p, proteins known to cooperatively activate transcription of Ty1 elements and the TEC1 gene during development of pseudohyphae. Yeast with a deletion of STE12 does not express FLO11 transcripts, indicating that STE12 is required for FLO11 expression. These ste12-deletion strains also do not invade agar. However, the ability to invade can be restored by overexpressing FLO11. Activation of FLO11 may thus be the primary means by which Ste12p and Tec1p cause invasive growth.

Molecular recognition of pathogen attack occurs inside of plant cells in plant disease resistance specified by the Arabidopsis genes RPS2 and RPM1

The Arabidopsis thaliana disease resistance genes RPS2 and RPM1 belong to a class of plant disease resistance genes that encode proteins that contain an N-terminal tripartite nucleotide binding site (NBS) and a C- terminal tandem array of leucine-rich repeats. RPS2 and RPM1 confer resistance to strains of the bacterial phytopathogen Pseudomonas syringae carrying the avirulence genes avrRpt2 and avrB, respectively. In these gene-for-gene relationships, it has been proposed that pathogen avirulence genes generate specific ligands that are recognized by cognate receptors encoded by the corresponding plant resistance genes. To test this hypothesis, it is crucial to know the site of the potential molecular recognition. Mutational analysis of RPS2 protein and in vitro translation/translocation studies indicated that RPS2 protein is localized in the plant cytoplasm. To determine whether avirulence gene products themselves are the ligands for resistance proteins, we expressed the avrRpt2 and avrB genes directly in plant cells using a novel quantitative transient expression assay, and found that expression of avrRpt2 and avrB elicited a resistance response in plants carrying the corresponding resistance genes. This observation indicates that no bacterial factors other than the avirulence gene products are required for the specific resistance response as long as the avirulence gene products are correctly localized. We propose that molecular recognition of P. syringae in RPS2- and RPM1-specified resistance occurs inside of plant cells.

Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family

The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance. We have cloned a transmembrane semaphorin, Sema W, that belongs to the class IV subgroup of the semaphorin family. The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form. The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs that we sequenced. Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but was found to be expressed at high levels in the adult central nervous system and lung. Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid showed that Sema W has growth-cone collapse activity against retinal ganglion-cell axons, indicating that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones. Genetic mapping of human SEMAW with human/hamster radiation hybrids localized the gene to chromosome 2p13. Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localized the gene to chromosome 6, and physical mapping placed the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189. This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease.

ARF-GEP100, a guanine nucleotide-exchange protein for ADP-ribosylation factor 6

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP100 activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP100, a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP100 in those areas. No similar coincidence of ARF-GEP100 with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.