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The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.

The phosphoinositide phosphatase SopB/SigD is a type III secretion system effector that plays multiple roles in Salmonella internalization and intracellular survival. We previously reported that SopB complexed with and inhibited the small GTPase Cdc42 when expressed in a yeast model system, independently of its phosphatase activity. Here we show that human Cdc42, but not Rac1, interacts with catalytically inactive SopB when coexpressed in Saccharomyces cerevisiae. This interaction occurs with both constitutively active and non-activatable Cdc42, suggesting that SopB binds Cdc42 independently of its activation state. By mutational analysis we have narrowed the Cdc42-interacting region of SopB to the first 142 amino acids, and isolated a collection of point mutations in this region, mainly affecting leucine residues conserved in the related Shigella IpgD protein. Such mutations yielded SopB unable to interact with Cdc42 but maintained phosphatase activity. SopB mutant proteins defective for binding Cdc42 were ubiquitinated upon translocation in mammalian cells, but their localization to the Salmonella-containing vacuole was reduced compared with wild-type SopB. Whereas invasion of mammalian cells by Salmonella bearing these sopB mutations was not affected, intracellular replication was less efficient, suggesting that SopB-Cdc42 interaction contributes to the adaptation of Salmonella to the intracellular environment.

Infection of cells and tissues by pathogenic microorganisms often involves severe reprogramming of host cell signaling. Typically, invasive microorganisms manipulate host cellular pathways seeking advantage for replication and survival within the host, or to evade the immune response. Understanding such subversion of the host cell by intracellular pathogens at a molecular level is the key to possible preventive and therapeutic interventions on infectious diseases. Reverse Protein Arrays (RPAs) have been exploited in other fields, especially in molecular oncology. However, this technology has not been applied yet to the study of infectious diseases. Coupling classic in vitro infection techniques used by cellular microbiologists to proteomic approaches such as RPA analysis should provide a wealth of information about how host cell pathways are manipulated by pathogens. The increasing availability of antibodies specific for phosphorylated epitopes in signaling proteins allows monitoring global changes in phosphorylation through the infection process by utilizing RPA analyses. In our lab, we have shown the potential of RPA technology in this field by devising a microarray consisting of lysates from cell cultures infected by Salmonella typhimurium. The protocols used are described here.

Infection of cells and tissues by pathogenic microorganisms often involves severe reprogramming of host cell signaling. Typically, invasive microorganisms manipulate host cellular pathways seeking advantage for replication and survival within the host, or to evade the immune response. Understanding such subversion of the host cell by intracellular pathogens at a molecular level is the key to possible preventive and therapeutic interventions on infectious diseases. Reverse Protein Arrays (RPAs) have been exploited in other fields, especially in molecular oncology. However, this technology has not been applied yet to the study of infectious diseases. Coupling classic in vitro infection techniques used by cellular microbiologists to proteomic approaches such as RPA analysis should provide a wealth of information about how host cell pathways are manipulated by pathogens. The increasing availability of antibodies specific for phosphorylated epitopes in signaling proteins allows monitoring global changes in phosphorylation through the infection process by utilizing RPA analyses. In our lab, we have shown the potential of RPA technology in this field by devising a microarray consisting of lysates from cell cultures infected by Salmonella typhimurium. The protocols used are described here.

This paper presents a study on the deformation of anisotropic fibrous porous media subjected to moistening by water in the liquid phase. The deformation of the medium is studied by applying the concept of effective stress. Given the structure of the medium, the displacement of the solid matrix is not taken into account with respect to the displacement of the liquid phase. The transport equations are derived from the model proposed by Narasimhan. The transport coefficients and the relation between the variation in apparent density and effective stress are obtained by test measurements. A numerical model has been established and applied for studying drip moistening of mineral wool samples capable or incapable of deformation.

New 3-hydrazino-5,6-dihydrobenzo[h]cinnolines were prepared, and their effects on arterial pressure, diuresis and Na+ and K+ excretion in conscious normotensive rats were studied.

The hydromechanical behavior of a moist fibrous medium is tested with the help of the principle of attenuation of two collinear gamma rays (cesium and americium). The detector is made up of a single spectrometric acquisition system and of a multichannel analyzer which differentiates the two gamma rays. This differentiation allows the simultaneous discovery of the density and of the moisture at each part of the medium. The measurement is carried out by moving the sample tested in relation to the gamma ray with the help of a very accurate apparatus which functions with a stepping motor engine. A microcomputer carries out the positioning, the measurement findings, and the signal processing. Software programs have been developed which define the parameters of each experiment as well as visualizing the diffusion and deformation properties of the medium tested. Numerical simulations representative of the medium in experimental situation are presented. This technique, believed original, permitted the study of the hydrodynamical behavior of a macroporous medium (containing 98% of air) in a way unused so far.

Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island-1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re-programming host cell pathways, we used ZeptoMARK reverse-phase protein array technology, which allows printing 32-sample lysate arrays that can be analyzed with phospho-specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS-deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen-activated protein kinases, mainly dependent on SopE/E2, as well as SigD-dependent phosphorylation of PKB/Akt and its targets GSK-3beta and FKHR/FoxO. This is the first time that reverse-phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.

In animal cells, Akt (also called protein kinase B) is activated by stimuli that elevate the level of phosphatidylinositol 3,4,5-trisphosphate and is a major effector for eliciting responses that support cell growth and survival. We have shown previously that co-expression of Akt1 in budding yeast (Saccharomyces cerevisiae) along with hyperactive p110α, the catalytic subunit of mammalian phosphatidylinositol 3-kinase, results in Akt1 relocalization to cellular membranes and activation. In the present study, we show that activation of all three mammalian Akt isoforms by wild-type p110α causes deleterious effects on yeast cell growth. Toxicity of Akt in S. cerevisiae required its catalytic activity, its pleckstrin homology domain, and phosphorylation of its activation loop, but not phosphorylation of its hydrophobic motif. We demonstrate that expression in yeast of the only purported oncogenic allele, Akt1(E17K), leads to enhanced phenotypes. Ala-scanning mutagenesis of the VL1 region within the phosphatidylinositol 3,4,5-trisphosphate-interacting pocket of the Akt1 pleckstrin homology domain revealed that most residues in this region are essential for Akt1 activity. We found that active Akt leads to enhanced signaling through the yeast cell wall integrity pathway. This effect requires the upstream Rho1 activator Rom2 and involves both phosphorylation of the MAPK Slt2 and expression of its transcriptional targets, thus providing a quantitative reporter system for heterologous Akt activity in vivo. Collectively, our results disclose a heterologous yeast system that allows the functional assessment in vivo of both loss-of-function and tumorigenic Akt alleles.

Nail biopsy is thought to be a useful technique for the diagnosis of diseases affecting the nail apparatus and may help avoid delays in the diagnosis of important mucocutaneous diseases. Furthermore, it has therapeutic value in its own right. It is not a difficult technique to perform but it requires an in-depth knowledge of the anatomy and physiology of the nail unit, as well as surgical experience and patient collaboration. In order to assess the diagnostic utility of this technique, we reviewed the nail biopsies performed in our department between June 2005 and May 2006.We identified 15 patients in whom nail biopsy had been performed. The clinical findings, type of biopsy performed, and histopathologic diagnosis were assessed.Nail biopsy allowed diagnosis of a variety of skin disease in 13 out of 15 patients (psoriasis in 5, onychomycosis in 4, melanonychia in 2, melanoma in 1, and subungual hematoma in 1). None of the patients presented sequelae as a result of the intervention after several months of follow-up.Nail biopsy is a useful tool in cases in which the patient history, clinical presentation, and additional tests have not led to a definitive diagnosis. In our experience, it can be performed safely and with minimal scarring.

Glass fiber insulant is a medium which can be deformed when loaded. Moisture transfer can occur in it. An experimental installation has been de signed in order to determine the water content and the deformation. The basic idea is the principle of attenuation of two radioactive emissions As a first approach, the measures have been carried out with media held rigidly. They show how the fiber di ameter and nominal density act upon the coefficients of moisture transfer and the properties of capillarity of glass fiber insulant

In Saccharomyces cerevisiae, a variety of stresses and aggressions to the cell wall stimulate the activation of the cell wall integrity MAPK pathway, which triggers the expression of a series of genes important for the maintenance of cell wall homeostasis. This MAPK module lies downstream of the Rho1 small GTPase and protein kinase C Pkc1 and consists of MAPKKK Bck1, MAPKKs Mkk1 and Mkk2, and the Slt2 MAPK. In agreement with previous reports suggesting that Mkk1 and Mkk2 were functionally redundant, we show here that both Mkk1 and Mkk2 alone or even chimerical proteins constructed by interchanging their catalytic and regulatory domains are able to efficiently maintain signal transduction through the pathway. Both Mkk1 and Mkk2 are phosphorylated in vivo concomitant to activation of the cell integrity pathway. Interestingly, hyperphosphorylation of the MEKs required not only the upstream components of the pathway, but also a catalytically competent Slt2 MAPK downstream. Active Slt2 purified from yeast extracts was able to phosphorylate Mkk1 and Mkk2 in vitro. We have mapped Ser(50) as a direct phosphorylation target for Slt2 in Mkk2. However, substitution of all (Ser/Thr)-Pro canonical MAPK target sites with alanine did not totally abrogate Slt2-dependent Mkk2 phosphorylation. Mutation or deletion of a conserved MAPK-docking site at the N-terminal extension of Mkk2 precluded its interaction with Slt2 and negatively affected retrophosphorylation. Our data show that the cell wall integrity MAPKKs are targets for their downstream MAPK, suggesting the existence of complex feedback regulatory mechanisms at this level.

The signaling pathways involving class I phosphatidylinositol 3-kinases (PI3K) and the phosphatidylinositol-(3,4,5)-trisphosphate phosphatase PTEN regulate cell proliferation and survival. Thus, mutations in the corresponding genes are associated to a wide variety of human tumors. Heterologous expression of hyperactive forms of mammalian p110α and p110β in Saccharomyces cerevisiae leads to growth arrest, which is counterbalanced by coexpression of mammalian PTEN. Using this in vivo yeast-based system, we have done an extensive functional analysis of germ-line and somatic human PTEN mutations, as well as a directed mutational analysis of discrete PTEN functional domains. A distinctive penetrance of the PTEN rescue phenotype was observed depending on the levels of PTEN expression in yeast and on the combinations of the inactivating PTEN mutations and the activating p110α or p110β mutations analyzed, which may reflect pathologic differences found in tumors with distinct alterations at the p110 and PTEN genes or proteins. We also define the minimum length of the PTEN protein required for stability and function in vivo. In addition, a random mutagenesis screen on PTEN based on this system allowed both the reisolation of known clinically relevant PTEN mutants and the identification of novel PTEN loss-of-function mutations, which were validated in mammalian cells. Our results show that the PI3K/PTEN yeast-based system is a sensitive tool to test in vivo the pathologic properties and the functionality of mutations in the human p110 proto-oncogenes and the PTEN tumor suppressor and provide a framework for comprehensive functional studies of these tumor-related enzymes. [Cancer Res 2007;67(20):9731–9]

A rate control (RC) algorithm for H.264 video coding with stored-B (SB) pictures is proposed for low-delay applications. Different models for P and SB pictures are defined for a better QP and MAD estimation. Furthermore, a novel saw-tooth shaped model of target buffer level has also been introduced for a proper bit allocation in GOP structures with SB pictures. Our experimental results show that this proposal outperforms the reference software RC in terms of buffer occupancy and target bit rate adjustment at the expense of slight quality reduction. Publicado

PO-191 Patient population with Multiple Myeloma and transitions across lines of therapy in the United States: an epidemiologic model J. Cid Ruzafa, E. Merinopoulou, R.F. Baggaley, P. Leighton, W. Werther, D. Felici, A. Cox Retrospective Observational Studies, Evidera, London, UK: JCR, EM, PL, AC; Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, UK: RFB; Onyx Pharmaceuticals, an Amgen Subsidiary, San Francisco, CA, USA: WW, DF

e13523 Background: The phosphatidylinositol 3-kinase (PI3K) pathway couples receptor-mediated signaling to essential cellular functions by generating the lipid second messenger phosphatidylinositol...

Heterologous expression of bacterial virulence factors inSaccharomyces cerevisiaeis a feasible approach to study their molecular function. The authors have previously reported that theSalmonella typhimuriumSigD protein, a phosphatidylinositol phosphatase involved in invasion of the host cell, inhibits yeast growth, presumably by depleting an essential pool of phosphatidylinositol 4,5-bisphosphate, and also that a catalytically inactive version, SigDR468A, was able to arrest growth by a different mechanism that involved disruption of the actin cytoskeleton. This paper describes marked differences between the phenotypes elicited by expression of SigD and SigDR468Ain yeast. First, expression of SigDR468Acaused accumulation of large unbudded cells and loss of septin organization, while SigD expression caused none of these effects. Second, growth inhibition by SigDR468Awas mediated by a cell cycle arrest in G2 dependent on the Swe1 morphogenetic checkpoint, but SigD-induced growth inhibition was cell cycle independent. And third, SigD caused strong activation of the yeast MAP kinase Slt2, whereas SigDR468Arather inactivated another MAP kinase, Kss1. In a screen for suppressors of SigDR468A-induced growth arrest by overexpression of a yeast cDNA library, the Cdc42 GTPase was isolated. Furthermore, SigDR468Awas co-purified with Cdc42 from yeast lysates. It is concluded that theSalmonellaSigD protein deprived of its phosphatase activity is able to disrupt yeast morphogenesis by interfering with Cdc42 function, opening the possibility that the SigD N-terminal region might directly modulate small GTPases from the host during infection.

The design of structures and algorithms for non-MAP multiclass decision problems is discussed in this paper. We propose a parametric family of loss functions that provide the most accurate estimates for the posterior class probabilities near the decision regions. Moreover, we discuss learning algorithms based on the stochastic gradient minimization of these loss functions. We show that these algorithms behave like sample selectors: samples near the decision regions are the most relevant during learning. Experimental results on some real datasets are also provided to show the effectiveness of this approach versus the classical cross entropy (based on a global posterior probability estimation).

Mitotic yeast (Saccharomyces cerevisiae) cells express five related septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) that form a cortical filamentous collar at the mother-bud neck necessary for normal morphogenesis and cytokinesis. All five possess an N-terminal GTPase domain and, except for Cdc10, a C-terminal extension (CTE) containing a predicted coiled coil. Here, we show that the CTEs of Cdc3 and Cdc12 are essential for their association and for the function of both septins in vivo. Cdc10 interacts with a Cdc3–Cdc12 complex independently of the CTE of either protein. In contrast to Cdc3 and Cdc12, the Cdc11 CTE, which recruits the nonessential septin Shs1, is dispensable for its function in vivo. In addition, Cdc11 forms a stoichiometric complex with Cdc12, independent of its CTE. Reconstitution of various multiseptin complexes and electron microscopic analysis reveal that Cdc3, Cdc11, and Cdc12 are all necessary and sufficient for septin filament formation, and presence of Cdc10 causes filament pairing. These data provide novel insights about the connectivity among the five individual septins in functional septin heteropentamers and the organization of septin filaments.

Most studies focus on the ecotoxicity of pulp and paper mill effluents, rather than on how they affect the physicochemical and biological structure and the intrinsic ecological capabilities of the receiving watercourses. We investigated the impact of such effluents on the water quality, microplankton system and microbial self-purification capacity (degradation of polymeric organic compounds via extracellular enzymes) of the Biobio River in Chile. The physicochemical impact on the water quality was indicated by raised conductivity, by the pollution of the water body with nitrate, nitrite and soluble reactive phosphorus, by the appearance of tannin and lignin, and by the steady accumulation of inorganic and organic suspended matter (SPM) along the river. From the biological structure of the microplankton system, very low and declining concentrations of chlorophyll a and heterotrophic flagellate densities were determined. The pulp and paper mill effluents introduced high bacterial abundances and biomass concentrations into the river water. This reflects the effective use made of the abundantly available inorganic and organic nutrients within this industrial and municipal process water by bacteria adapted to these extreme environments, additionally supported by concomitant low grazing pressure derivable from low heterotrophic flagellate abundances. Indeed, in one section of the river affected by a pulp mill, the plant was found to significantly contribute to the self-cleaning capacity of the river. However, this elevated degradation capacity was not enough to compensate for the additionally discharged organic material which, together with the toxic effects of the paper plant effluents, significantly interferes with the ecological status of the Biobio River.

A CAD approach permitting us an easy frequency response simulation of LTI systems implemented with analog electronic-devices, is introduced. This technique uses the features of symbolic analysis in order to get more insight into the behavior of the circuit. An important thing is that a reasonable amount of computer effort is saved through modeling several analog circuits using nullors. Most important is that using nullors, a pure nodal-analysis method is established in order to improve the computation of transfer functions. The paper also demonstrates the suitability and appropriateness of the proposed simulation technique to be used in education.

A simple low-cost FI-FAAS methodology was developed for determination of Cd traces in food samples using preconcentration by on-line extraction in a chelating resin, a strategy usually applied for analytical measurements in water samples. In the case of food samples, a more complex matrix medium, the pH of acid digested solutions (pH 1) was raised first to pH 3-5 off-line with 0.5 mol/L NH3, and then was on-line adjusted to pH 8.0 with ammonium acetate buffer (1.15 mol/L CH3COOH / 2.0 mol/L NH3) to proper retention of Cd as Cd-8-hydroxiquinoline complex, minimizing interferences. Cadmium was preconcentrated in a minicolumn filled with 8-hydroxiquinoline azo-immobilized on controlled-pore glass (80 mg). A multivariate approach was adopted to establish the main parameters involved in the FI system. Subsequently, the sample, eluent, and buffer flow rates were optimized using an univariate approach. An enrichment factor of 27 was achieved for 3.0 mL of reference solution and the sampling frequency was 63 h-1. The detection limit (3sBL / slope) was 0.7 and 0.4 mg/L for a preconcentration time of 30 and 60 s, respectively. The developed methodology was validated employing certified reference materials (Oyster tissue, Mussel, and Spiked skim milk powder) and the determined and certified values are in agreement (95% confidence level). The methodology was applied for trace levels determination of Cd in lyophilized mussel samples from the Chilean coast and non-fat milk powder, and a comparison made with graphite furnace electrothermal atomization (ETAAS) showed no statistically significant differences in results (95% confidence level) Se desarrolló una metodología simple y de bajo costo por EAALL-IF para la determinación de trazas de Cd en muestras de alimentos empleando preconcentración por extracción en línea con una resina quelante, una estrategia aplicada normalmente en mediciones analíticas realizadas en muestras de agua. En muestras de alimentos, siendo esta una matriz más compleja, el pH de las soluciones de digeridos ácidos (pH 1) fue aumentado primero fuera de línea hasta pH 35 con 0.5 mol/L NH3, y luego, en el sistema en línea se ajustó a pH 8.0 con tampón de acetato de amonio (1.15 mol/L CH3COOH / 2.0 mol/L NH3) para una retención apropiada de Cd como complejo Cd-8-hidroxiquinoleina minimizando las interferencias. El cadmio fue preconcentrado en una minicolumna rellena con 8-hidroxiquinoleina azo-inmovilizada sobre perla de vidrio de poro controlado (80 mg). Se adoptó un procedimiento multivariado a fin de establecer los principales parámetros involucrados en el sistema de IF. Posteriormente, se ajustaron los flujos de la solución muestra, el eluente, y el tampón empleando un procedimiento univariado. Se logró un factor de enriquecimiento de 27 para 3.0 mL de solución de referencia y la frecuencia de muestras fue 63 h-1. El límite de detección (3sBL / pendiente) correspondió a 0.7 y 0.4 mg/L para un tiempo de preconcentración de 30 y 60 s, respectivamente. La metodología fue validada empleando materiales de referencia certificados (tejido de ostras, moluscos, y leche en polvo desnatada con recarga de Cd) obteniéndose resultados concordantes con los valores certificados (95% de nivel de confianza). Se aplicó en la determinación de trazas de Cd en muestras de moluscos liofilizados obtenidos en la costa chilena y de leche en polvo descremada, y una comparación efectuada con atomización electrotérmica por horno de grafito (ETAAS) no presentó diferencias estadísticamente significativas en los resultados (95% de nivel de confianza)

Seismic responses of different sites of Barcelona have been investigated through numerical modelling. Geological maps and geotechnical data available from drillings for buildings and infrastructures have been used to determine the dynamical properties of the soils through different correlations between standard geotechnical data and dynamical parameters obtained in other regions. An estimation of the depth of the Palaeozoic basement has been obtained through an inversion of a detailed gravity survey. A 1-D equivalent linear method has been used to compute complete transfer functions and other spectral responses, such as PSA and PSV for various damping values, with the purpose of classifying zones with similar behaviour. Given the uncertainties associated with the input data, a Montecarlo’s simulation process has been carried out. Four zones, characterized by their corresponding transfer function and by PGA amplifications, are proposed. The numerical results are compared with those previously obtained through microtremor measurements, showing that predominant periods derived from Nakamura’s technique should be taken carefully.

The Saccharomyces cerevisiae CDC15, DBF2, TEM1 and CDC14 genes encode regulatory proteins that play a crucial role in the latest stages of the M phase of the cell cycle. By complementation of a S. cerevisiae cdc15-lyt1 mutant with a Candida albicans centromeric-based genomic library, we have isolated a homologue of the protein phosphatase-encoding gene CDC14. The sequence analysis of the C. albicans CDC14 gene reveals a putative open reading frame of 1626 base pairs interrupted by an intron located close to the 5' region. Analysis of C. albicans cDNA proved that the intron is processed in vivo. The CaCDC14 gene shares 49% of amino acid sequence identity with the S. cerevisiae CDC14 gene, 46% with Schizosaccharomyces pombe homologue, 35% with Caenorhabditis elegans and 37% and 38% with human CDC14A and CDC14B genes, respectively. As expected, the C. albicans CDC14 gene complemented a S. cerevisiae cdc14-1 mutant. We found that this gene was able to efficiently suppress not only a S.cerevisiae cdc15-lyt1 mutant but also a dbf2-2 mutant in a low number of copies and allowed growth, although very slightly, of a tem1 deletant. Overexpression of the human CDC14A and CDC14B genes complemented, although very poorly, S. cerevisiae cdc15-lyt1 and dbf2-2 mutants, suggesting a conserved function of these genes throughout phylogeny. The sequence of CaCDC14 was deposited in the EMBL database under Accession No. AJ243449.

This is an open access article distributed under the Creative Commons Attribution License.-- et al., Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of β1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect β1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation., The work described in this article was part of the EUROFAN II program funded by the European Union and co-financed by grants BIO99-1384-CE and BIOg8-1516-CE from Comisión Interministerial de Ciencia y Tecnología (CICYT, Spain).

The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial beta-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Delta crh2Delta strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1-green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle.

Exit from mitosis in the budding yeast Saccharomyces cerevisiae cell cycle is regulated by a regulatory network that involves, among other proteins, the small GTPase Tem1, the protein phosphatase Cdc14, and the protein kinases Dbf2 and Cdc15. Using a fusion to jellyfish green fluorescent protein (GFP), here we report that Cdc15 costains with the microtubular-organizing apparatus and that this localization is precluded in a mutant lacking the outer plaque of the spindle pole body (SPB). The appearance of Cdc15 in the SPB is asymmetric and cell-cycle-regulated, preferentially marking the daughter cell SPB at anaphase and eventually disappearing at cytokinesis. Overproduction of GFP-tagged Cdc15 led to an accumulation of the fusion protein in both mother and daughter cells SPBs and, transiently, in small budded cells and shmoos. The Cdc15 localization pattern was maintained in dbf2, cdc14 and anaphase-promoting complex (cdc16) mutants, suggesting that the function of these proteins is not related to the localization of Cdc15 to the SPB but rather, at least in the case of Cdc14, to its timely removal from this structure. Tem1-depleted cells kept alive by Cdc15-GFP overexpression still display a proper localization of Cdc15. The results presented here suggest that the transient cell-cycle-dependent localization of Cdc15 to the SPB plays a role in the regulation of the latest stages of the cell cycle.

Evidence-based public health can be viewed as a way of facing community health problems by which the decisions are taken accordingly to the available evidence. A complete multidisciplinary approach should be introduced, with the goal of identifying and articulating the different dimensions which are present when trying to respond to the challenges or problems for the people's health. In addition to clinical medicine, disciplines such as epidemiology, biostatistics, health services research, economics, behavioural sciences, demography, nutrition, gerontology or computing sciences, among others, may contribute to health promotion and disease prevention. This open approach should not be exempt of a captious attitude toward the obtainable information, while it is essential to have a comprehensive spirit to combine this information with the previous experience and the peculiarities of each situation. Searching for evidence begins with a clear operational statement of the problem. This helps to select and obtain the key pieces of information. The information obtained must be judged critically, and related to the specifics of the problem. Lastly, it should be accepted that search for evidence does not mean finding certitude. Idolizing evidence can end up being as inappropriate as the implementation of a health program ignoring the previous information about its potential effectiveness, if such information were available. Many decisions in public health have to be taken conscious that scientific evidence does not rule out uncertainty, as other elements such as the cultural context, the individual preferences and other social factors may have a decisive influence in the success or failure of public health interventions.

We have evaluated in vitro the effects of using plasma (group A) or PAS-2 (group B) in storing platelets, regarding as well the type of container (conventional polyolefin--PL732--or new oxygen permeable platelet containers--PL2410, Compoflex--), on the metabolism of platelet concentrates--PC--from pools of five buffy coats.87, A and B pools of PCs collected in two types of bags were studied. The samples were taken on days 2, 5 and 7, and cell counts, pH, glucose, platelet activation (% CD62) and aggregation response were measured.In group A, when we compared the two types of bags, we observed a difference (p0.01) in values of pH and glucose on day 5. These values were more advantageous in new oxygen permeable platelet containers. The rest of the parameters analysed didn't show significant differences. When we compared the PL-2410 containers from groups A and B, we found a lower level of glucose (p0.01) in group B, although the levels of glucose in this group on days 5 and 7 of storage were sufficient to support the platelet metabolism.The use of new oxygen permeable polyolefin containers and additive solutions, PAS-2, allows us to obtain pools of PC with suitable metabolic parameters during storage.

A flow-injection (FI) system with a minicolumn of bonded silica with octadecyl groups (C-18) to collect diethyldithiocarbamate complexes of Cd and Pb in reference solutions and in acid-digested hair and blood solutions was developed and evaluated by flame atomic absorption spectrometry (FAAS). The system was optimized by multivariate method, based on a factorial experimental design in two levels, selecting eight parameters that mostly affected the expected analytical signal. The detection limits (3×sBL/slope, 60 s preconcentration) were 0.7 and 5 µg/l for Cd and Pb, respectively and the sampling frequency was 40 samples/h. Effects of interfering ions are discussed. The methodology was validated by analysis of certified reference materials of hair and blood for Pb, and by recoveries of Cd and Pb spikes performed in hair and blood samples. Results for Pb agreed well with certified values and recoveries were satisfactory (103% in blood and 100% in hair). Also, the recovery of Cd in hair was fair (107%); however, in blood it was not quantitative (22%) Se desarrolló y evaluó por espectrometría de absorción atómica con llama (EAALL) un sistema de inyección en flujo (IF) con una mini columna rellena con gel de sílice enlazada con grupos octadecilo (C-18) para retener complejos de dietilditiocarbamatos de Cd y Pb en soluciones de referencia y de digestos ácidos de cabello y sangre. El sistema fue optimizado por método multivariado en base a un diseño experimental factorial en dos niveles, seleccionándose los ocho parámetros que influyen más significativamente en la señal de analito esperada. Los límites de detección 3×sBL/pendiente, con 60 seg de preconcentración) fueron 0.7 y 5 µg/L para Cd y Pb, respectivamente y la frecuencia de muestreo fue 40 muestras/hr. Se discuten los efectos de iones interferentes. La metodología fue validada para Pb por análisis de materiales de referencia certificados de cabello y sangre y por estudios de recuperación de Cd y Pb efectuados en muestras de cabello y sangre. Los resultados obtenidos para Pb son concordantes con las concentraciones certificadas y fueron satisfactorios en los estudios de recuperación (103% en sangre y 100% en cabello). También, la recuperación de Cd en cabello fue adecuada (107%); sin embargo, en sangre no fue cuantitativa (22%)

A series of novel conformationally restricted butyrophenones (2-(aminoethyl)- and 3-(aminomethyl)thieno- or benzocycloalkanones bearing (6-fluorobenzisoxazolyl)piperidine, (p-fluorobenzoyl)piperidine, (o-methoxyphenyl)piperazine, or linear butyrophenone fragments) were prepared and evaluated as atypical antipsychotic agents by in vitro assays of affinity for dopamine receptors (D(1), D(2)) and serotonin receptors (5-HT(2A), 5-HT(2C)) and by in vivo assays of antipsychotic potential and the risk of inducing extrapyramidal side effects. Potency and selectivity depended mainly on the amine fragment connected to the cycloalkanone structure. As a group, compounds with a benzisoxazolyl fragment had the highest 5-HT(2A) activities, followed by the benzoylpiperidine derivatives; in general, alpha-substituted cycloalkanone derivatives were more active than the corresponding beta-substituted congeners. CoMFA (comparative molecular field analysis) and docking studies showed electrostatic, steric, and lipophilic determinants of 5-HT(2A) and D(2) affinities and 5-HT(2A)/D(2) selectivity. The in vitro and in vivo pharmacological profiles of N-[(4-oxo-4H-5, 6-dihydrocyclopenta[b]thiophene-5-yl)ethyl]-4-(6-fluorobenzisox azol-3 -yl)piperidine (23b, QF 0510B), N-[(4-oxo-4,5,6, 7-tetrahydrobenzo[b]thiophene-5-yl)ethyl]-4-(6-fluorobenzisoxazol- 3-y l)piperidine (24b, QF 0610B), and N-[(7-oxo-4,5,6, 7-tetrahydrobenzo[b]thiophene-6-yl)ethyl]-4-(6-fluorobenzisoxazol- 3-y l)piperidine (29b, QF 0902B) suggest that they may be effective antipsychotic drugs with low propensity to induce extrapyramidal side effects.

The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37 degrees C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.

To describe the reported usual dietary intakes of the participants in the Salisbury Eye Evaluation (SEE) project and to estimate the prevalence of inadequate nutrient intakes using the probability approach.A representative sample of elderly residents (aged 65 to 85 years) of Salisbury, Md.Cross-sectional survey, using a food frequency questionnaire to obtain nutrient intakes. We estimated energy and protein; percent of energy intake from carbohydrates, fat, and protein; as well as usual intakes of cholesterol, vitamin A, carotenoids, vitamin C, thiamin, riboflavin, vitamin B-6, vitamin E, niacin, iron, calcium, zinc, and folate. Estimates of prevalence of inadequate nutrient intakes were calculated using the probability approach among the 2,655 participants with complete nutrient intake information.The chi 2 test for independence and analysis of variance. A P.05 was considered significant in a 2-sided test.On average, white participants of both genders reported higher mean energy and nutrient intakes than did black participants. Zinc had the highest estimated prevalences of inadequacy across all gender and race categories, followed by calcium, vitamin E, and vitamin B-6. Vitamin C, with estimated prevalences of inadequacy lower than 13%, and folate, with prevalences lower than 17%, had the lowest estimated prevalences of inadequacy across all gender, race, and age categories.In this population, there are race differences in estimated prevalences of inadequate nutrient intake. According to the current nutrient requirements for adults aged 51 years and older, many elderly persons have inadequate dietary intakes of key nutrients.

Summary: The non-sporulating diploid strain V327 of Saccharomyces cerevisiae was previously isolated in a search for thermosensitive autolytic mutants. This strain is very efficient at releasing intracellular proteins into the medium when incubated at high temperatures. The expression of this lytic phenotype depends on a morphogenetic defect, consisting of the appearance of elongated chains of cells. Transmission electron microscopy revealed a mislocalization of septa at semi-permissive temperatures and a total lack of septation together with abnormal cell wall architecture at a non-permissive temperature. The septin-encoding CDC10 gene was cloned by complementation of the pleiotropic phenotype of the V327 mutant. Rescue and sequencing of CDC10 alleles from V327 revealed a point mutation that created a single amino acid change in a region which is well conserved among septins. This new allele was named cdc10-11. The construction of a cdc10-11 haploid strain by substituting the CDC10 gene with the rescued allele permitted further genetic analyses of the mutation and allowed the construction of new homozygous cdc10-11 diploid strains that showed a reduced ability to sporulate. Fusing both the wild-type and the cdc10-11 alleles to green fluorescent protein (GFP) demonstrated that the mutation does not affect the localization of this septin to the bud neck at the standard growth temperature of 24 °C, although the morphogenetic phenotype at 37 °C parallels the disappearance of Cdc10-GFP at the ring encircling the septum area.

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

We have deleted six different ORFs of unknown function located on the right arm of Saccharomyces cerevisiae chromosome VII; namely, YGR187c/HGH1, YGR189c, YGR194c, YGR195w, YGR196c and YGR198w. No basic phenotypes could be attributed to the strains deleted in any of genes YGR187c/HGH1, YGR189c, YGR194c and YGR196c. These deletants did not show mating, sporulation or growth defects under any of the conditions tested. However, spores bearing deletions in either the YGR195w or YGR198w genes were unable to develop into macroscopical colonies. The YGR195w gene product shows significant homology with bacterial ribonuclease PH, an enzyme hitherto undescribed in yeasts, and its deletion causes a loss of viability after one to three rounds of cell division. Overexpression of this gene, using a tetracycline-regulatable promoter system, did not cause any effect on the cells. Contrary to what has been reported for prokaryotic homologs, this enzyme could play an essential role in yeast cell biology. The product encoded by the other essential ORF, YGR198w, shows no significant homology with any protein of known function in the databases. Spores bearing the deletion usually germinate and give rise to microcolonies of 50-100 non-viable cells.

Two different techniques have been applied to evaluate the potential seismic site effects in the city of Barcelona. Transfer functions have been computed in different sites, through a unidimensional linear-equivalent model using the available dynamic parameters of soils obtained from drillings for building and infrastructure public works. The depth of the basement and the structural framework have been determined from the inverse solution of the residual gravity anomaly. Microtremor measurements using Nakamura's technique are also available for these sites. The results of these techniques have been compared. We prove that the predominant frequencies obtained by Nakamura's technique agree with the fundamental frequencies in the computed transfer functions, but for complex structures these fundamental frequencies are not the frequencies for which largest amplifications appear. In our case, the fundamental frequency is related with the post-Palaeozoic deposit thickness, which is defined by the results from inversion of a detailed gravity survey, while the maximum amplification depends more on the uppermost soft soils.

In Public Health exists a growing tendency to evaluate the impact of health problems both on the quality of life of the persons involved as well as the use of health services. In this sense, the evaluation of incapacity is acquiring ever greater relevance. The Barthel Index is an instrument widely used to this end and measures the capacity of the person for the execution of ten basic activities in daily life, obtaining a quantitative estimation of the subject's level of dependency. The Barthel Index has been used, since its introduction in 1955, resulting in numerous versions, as well as serving as a standard of comparison with other scales. It is an easily applicable method, with a high level of reliability and validity, capable of detecting changes, easy to interpret and the application of which is not problematic. On the other hand, its adaptation to different cultural environments is almost immediate. Although it has a few limitations, the Barthel Index may be recommended as a selection method for measuring physical incapacity, both in clinical practice as well as in epidemiological investigation and Public Health.

The classification of a data collection using tree structures has been studied by statisticians and psychologists for many years, and it has shown to be an effective way of dividing a complex classification problem in a sequence of simpler decision tasks. The modularity of both learning and classification showed by these structures has attracted the attention of neural network researchers, looking for alternatives to the learning and computational problems of backpropagation networks. This paper overviews the current research on tree classification based on neural networks. Structure and learning algorithms are described; the implications of probabilistic interpretations of the network behaviour are discussed and some new learning rules that can speed up learning and reduce the final misclassification probability are proposed.

The chemical and nutricional properties of flour from total calf omasum are assessed with the purpose of considering the utilization of a waste product from the meat industry as animal food. The results of the chemical percent analysis showed a high protein content of 78.6 g/100 g, a total lipid amount of 3.70 g/100 g and a cholesterol concentration of 0.14 g/100 g. From the study of the total saturated fatty acid composition it is deduced that palmitic acid prevails with a value of 28.4%. Unsaturated fatty acids show a value of 75% for oleic acid, this being the highest concentration. The values found for lipid composition are similar a fat bovine. Biological techniques were used to evaluate nitrogen utilization: Net Protein Utilization (NPU), true Digestibility (tD) and Biological Value (BV); a proof with casein as reference was simultaneously performed. The NPU, tD and the BV values found were: 58 +/- 7.0; 87 +/- 8.5 and 67 respectively. These results demonstrate that the assayed meat by-product had the adecuate nutritive elements for its industrialization as porcine balanced food.

The reactions which involve oxidants and free radicals species have played an essential role at the beginning of aerobic life, and they are an intrinsic part in the regulation of the cellular processes. However, as a result of the derived toxic effects of these oxidative processes, antioxidants molecules appeared in the very early stages of the evolution and they are able to control the production of these reactants and their dangerous effects. Oxidants and antioxidants have a clear function in the cellular physiology. When this delicate balance change many biochemical and cellular reactions are altered, and that may cause different pathologic diseases. In addition, free radicals of oxygen are thought to play a growing role in the biological process of ageing (1). It is not unusual to find papers about these reactants in every topic of the biomedicine. The main oxidants and free radicals in the organisms are oxygen-related agents, which are globally called reactive oxygen intermediates (ROI). These unavoidable, useful and dangerous biological oxidants are well characterized, although the recent implications they received in some pathologies deserve, in our opinion, a general review.

Receptors for the Fc fragment of IgG (Fc gamma R) have a well-documented role in the generation of oxidative burst. It is tempting to speculate that the type of interaction with Fc gamma R could be a mechanism of regulation of this process. Here we report on a comparative study of the induction of oxidative burst in human monocytes activated by means of different types of interaction with Fc gamma R. We studied non-primed monocytes obtained by centrifugal elutriation from healthy donors. These cells were submitted to Fc gamma R interactions following two distinct models: one, using particulate material (IgG-SRBC leading to phagocytosis or rosetting), and another using soluble reagents followed by cross-linking of the receptors (monoclonal antibodies against Fc gamma RI and Fc gamma RII and natural ligands, namely several isotypes of murine and human IgG). Phagocytosis and oxidative burst were studied simultaneously in the monocytes, following the methodology described recently. Human non-primed monocytes were able to generate a very obvious oxidative burst response after activation of Fc gamma R by particulate material. The same response was observed when Fc gamma RII was blocked by monoclonal antibodies. Ingestion was not necessary for activation of the oxidative burst, since the model of rosetting induced a level of burst generation similar to the one obtained in the phagocytic process. Cross-linking of Fc gamma RI by soluble reagents induced production of reactive oxidative intermediates (ROI) only when the ligand-binding site of the receptor was involved. These data lead to the conclusion that Fc gamma R interaction with soluble or particulate material induces oxidative burst in non-primed human monocytes only when the binding site of natural ligands is involved. The type of interaction also determines the efficiency of the generation of ROI. This fact could represent a regulatory mechanism.

In order to carry out a systematic search for mutants affected in cell integrity, the diploid strain Saccharomyces cerevisiae D1 was subjected to mutagenesis with ethyl methane sulphonate (EMS), and mutant clones were screened for thermosensitive autolytic phenotypes. The screening was based on examination of cell populations, from individual mutant clones, stained with propidium iodide to establish the proportion of cells lysing under non-permissive conditions by means of flow cytometry. Osmotic remediation of the autolytic phenotype in the presence of 1 M sorbitol was also checked. Out of 13,300 clones surviving mutagenesis, 34 were confirmed to be thermosensitive autolytic and 7 of them showed some osmotic complementation with regard to growth and cell lysis. The osmotic remediation in the other strains was negligible or affected only one of the two parameters. The expression of the mutant phenotype in the strains isolated led to a sporulation defect (40% of the strains) and significant alterations in morphology, such as cells in chains (35%), altered buds (25%) that eventually might elongate, round unbudded and highly vacuolated cells (12%) and large-sized cells (12%). These observations show that alterations in functions related to cell integrity can be correlated with an altered morphology. Genetic analysis of the mutant strains that could sporulate showed that in many instances the mutant phenotype was the result of more than one mutation, the mutations being individually recessive. However, at least one mutant strain, 933, carried a single mendelian mutation that was dominant in the diploid but haploid segregants were non-viable. Dominance of this mutation was also confirmed in tetraploids obtained by means of protoplast fusion.