SUBCELLULAR LOCALIZATION, PURIFICATION AND PROPERTIES OF A CDP-DIACYLGLYCEROL - DEPENDENT PHOSPHATIDYLSERINE SYNTHASE IN BACILLUS LICHENIFORMIS

A CDP-diacylglycerol dependent phosphatidylserine synthase was detected in three species of gram-positive bacilli, viz. Bacillus licheniformis, Bacillus subtilis and Bacillus megaterium; the enzyme in B. licheniformis was studied in detail. The subcellular distribution experiments in cell-free extracts of B. licheniformis using differential centrifugation, sucrose gradient centrifugation and detergent solubilization showed the phosphatidylserine synthase to be tightly associated with the membrane. The enzyme was shown to have an absolute requirement for divalent metal ion for activity with a strong preference for manganese. The enzyme activity was completely dependent upon the addition of CDP-diacylglycerol to the assay system; the role of the liponucleotide was rigorously shown to be that of phosphatidyl donor and not just a detergent-like stimulator. This enzyme was then solubilized from B. licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue-dextran Sepharose. The purified preparation showed a single band with an apparent minimum molecular weight of 53,000 when subjected to SDS polyacrylamide gel electrophoresis. The preparation was free of any phosphatidylglycerophosphate synthase, CDP-diacylglycerol hydrolase and phosphatidylserine hydrolase activities. The utilization of substrates and formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential BiBi reaction as opposed to the ping-pong mechanism exhibited by the well studied phosphatidylserine synthase of Escherichia coli. Proteolytic digestion of the enzyme yielded a smaller active form of the enzyme (41,000 daltons) which appears to be less prone to aggregation.^ This has been the first detailed study in a well-defined bacillus species of the enzyme catalyzing the CDP-diacylglycerol-dependent formation of phosphatidylserine; this reaction is the first committed step in the biosynthetic pathway to the major membrane component, phosphatidylethanolamine. Further study of this enzyme may lead to understanding of new mechanisms of phosphatidyl transfer and novel modes of control of phospholipid biosynthetic enzymes. ^

The localization and identification of candidate tumor suppressor genes involved in prostate cancer

Frequent loss of heterozygosity (LOH) at specific chromosomal regions are highly associated with the inactivation of tumor suppressor genes (TSGs) (Weinberg, 1991; Bishop, 1989). Chromosome 8p is the most frequently reported site of LOH (∼60%) in prostate cancer (PC), suggesting that there may be inactivated TSG(s) involved in PC on chromosome 8p. (Bergerheim et. al., 1991; Kagan et. al., 1995). In order to identify the smallest common regions of frequent LOH (SCLs) on chromosome 8, we screened 52 PC patient/tumor samples with 39 polymorphic markers in successive screenings. In the course of refining the SCLs, we identified 3 tumors with >6 Mb homozygous deletions (HZDs) at 8p22 and 8p21, suggesting the presence of candidate TSGs at both loci. These HZDs spanned the two SCLs at 8p22 (46%) and 8p21 (45%). The SCLs were narrowed to 3.2 cM at 8p22 and less than 3 cM at 8p21. ^ In order to identify candidate TSGs within the SCLs on 8p, two approaches were used. In the candidate gene approach, thirty genes that mapped to the SCLs were evaluated for expression in normal prostate and in PC cell lines. One of the candidate genes, Clusterin, showed decreased expression in 4/7 (57%) prostate cancer cell lines by Northern blot analysis. Clusterin will be further examined as a candidate TSG. ^ The second approach involved utilizing subtractive hybridization and hybrid affinity capture to generate pools of expressed sequence tags (ESTs) enriched for genes that are downregulated or deleted in PC and that map to specific regions of interest. We took advantage of a prostate cancer cell line (PC3) with a known HZD of a candidate TSG, CTNNA1 on 5q31, to develop and validate a model system. We then developed subtracted libraries enriched for 8p22 and 8p21 ESTs by this method, using two cell lines, MDAPCa-2b and PC3. The ESTs were cloned, and 40 were sequenced and evaluated for expression in normal prostate and PC cell lines. Three ESTs from the subtracted libraries, C2, C17 and F12, showed decreased expression in 29–57% of the prostate tumor cell lines studied, and will be further examined as candidate TSGs. ^

Mapping and characterization of the Xlsirt P11 RNA cis-acting localization element

In many organisms, polarity of the oocyte is established post-transcriptionally via subcellular RNA localization. Many RNAs are localized during oogenesis in Xenopus laevis, including Xlsirts ( Xenopus laevis short interspersed repeat transcripts) [Kloc, 1993]. Xlsirts constitute a large family defined by highly homologous repeat units 79–81 nucleotides in length. Endogenous Xlsirt RNAs use the METRO (Message Transport Organizer) pathway of localization, where RNAs are transported from the nucleus to the mitochondrial cloud in stage I oocytes. Secondly, RNAs anchor at the vegetal pole in stage II oocytes. Exogenous Xlsirt RNAs can also utilize the Late pathway of localization, which involves localization to the vegetal cortex during stage III of oogenesis and results in RNAs anchored in the cortex of the entire vegetal hemisphere. ^ The Xlsirts localization signal is contained within the repeat region. This study was designed to test the hypothesis that there are cis -acting localization elements in Xlsirts, and that higher order structure plays a role. Results of experiments on Xlsirt P11, a 1700 basepair (bp) family member, led to the conclusion that a 137-bp fragment of the repetitive region is necessary and sufficient for METRO and Late pathway localization. This analysis definitively demonstrates that the Xlsirt localization signal for the METRO and Late pathways reside within the repetitive region and not within the flanking regions. Analysis of Xlsirt linker scanning mutations revealed two METRO-pathway specific subelements, and one Late-pathway specific subelement. Functional, computer, and biochemical evidence relates the higher order structure of this element to its ability to function as a localization element. ^ Xlsirt 137 is 99% identical to the Xlsirt consensus sequence identified in this study, suggesting that it is the localization element for all localized Xlsirt family members. The repeat unit was reframed based on function, rather than arbitrarily based on sequence. This work supports the hypothesis presented in 1981 by George Spohr, who originally isolated the Xlsirts, which stated that the highly conserved repetitive elements must be constrained from variability due to some unknown function of the repeats themselves. These studies shed light on the mechanism of RNA localization, linking structure and function. ^

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^

Strike-slip faults mediate the rise of crustal-derived fluids and mud volcanism in the deep sea

We report on newly discovered mud volcanoes located at about 4500 m water depth 90 km west of the deformation front of the accretionary wedge of the Gulf of Cadiz, and thus outside of their typical geotectonic environment. Seismic data suggest that fluid flow is mediated by a >400-km-long strike-slip fault marking the transcurrent plate boundary between Africa and Eurasia. Geochemical data (Cl, B, Sr, 87Sr/86Sr, Delta18O, DeltaD) reveal that fluids originate in oceanic crust older than 140 Ma. On their rise to the surface, these fluids receive strong geochemical signals from recrystallization of Upper Jurassic carbonates and clay-mineral dehydration in younger terrigeneous units. At present, reports of mud volcanoes in similar deep-sea settings are rare, but given that the large area of transform-type plate boundaries has been barely investigated, such pathways of fluid discharge may provide an important, yet unappreciated link between the deeply buried oceanic crust and the deep ocean.

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na+/K+-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na+/K+-ATPase with the polyclonal antibody alpha (H-300) raised against the human alpha1-subunit of Na+/K+-ATPase. Na+/K+-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na+/K+-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na+/K+-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na+/K+-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 µmol/g FM/h) than in those of L. vulgaris (31.8 ± 3.3 µmol/g FM/h). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 µmol ATP/g FM/h, respectively. High concentrations of Na+/K+-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO2) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.

Nonparametric Message Passing Methods for Cooperative Localization and Tracking

The objective of this thesis is the development of cooperative localization and tracking algorithms using nonparametric message passing techniques. In contrast to the most well-known techniques, the goal is to estimate the posterior probability density function (PDF) of the position of each sensor. This problem can be solved using Bayesian approach, but it is intractable in general case. Nevertheless, the particle-based approximation (via nonparametric representation), and an appropriate factorization of the joint PDFs (using message passing methods), make Bayesian approach acceptable for inference in sensor networks. The well-known method for this problem, nonparametric belief propagation (NBP), can lead to inaccurate beliefs and possible non-convergence in loopy networks. Therefore, we propose four novel algorithms which alleviate these problems: nonparametric generalized belief propagation (NGBP) based on junction tree (NGBP-JT), NGBP based on pseudo-junction tree (NGBP-PJT), NBP based on spanning trees (NBP-ST), and uniformly-reweighted NBP (URW-NBP). We also extend NBP for cooperative localization in mobile networks. In contrast to the previous methods, we use an optional smoothing, provide a novel communication protocol, and increase the efficiency of the sampling techniques. Moreover, we propose novel algorithms for distributed tracking, in which the goal is to track the passive object which cannot locate itself. In particular, we develop distributed particle filtering (DPF) based on three asynchronous belief consensus (BC) algorithms: standard belief consensus (SBC), broadcast gossip (BG), and belief propagation (BP). Finally, the last part of this thesis includes the experimental analysis of some of the proposed algorithms, in which we found that the results based on real measurements are very similar with the results based on theoretical models.

Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana

Amidase 1 (AMI1) from Arabidopsis thaliana converts indole-3-acetamide (IAM), into indole-3-acetic acid (IAA). AMI1 is part of a small isogene family comprising seven members in A. thaliana encoding proteins which share a conserved glycine- and serine-rich amidase-signature. One member of this family has been characterized as an N-acylethanolamine-cleaving fatty acid amidohydrolase (FAAH) and two other members are part of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) or mitochondria (Tom complex) and presumably lack enzymatic activity. Among the hitherto characterized proteins of this family, AMI1 is the only member with indole-3-acetamide hydrolase activity, and IAM is the preferred substrate while N-acylethanolamines and oleamide are not hydrolyzed significantly, thus suggesting a role of AMI1 in auxin biosynthesis. Whereas the enzymatic function of AMI1 has been determined in vitro, the subcellular localization of the enzyme remained unclear. By using different GFP-fusion constructs and an A. thaliana transient expression system, we show a cytoplasmic localization of AMI1. In addition, RT-PCR and anti-amidase antisera were used to examine tissue specific expression of AMI1 at the transcriptional and translational level, respectively. AMI1-expression is strongest in places of highest IAA content in the plant. Thus, it is concluded that AMI1 may be involved in de novo IAA synthesis in A. thaliana.

Distributed collaborative processing in wireless sensor networks with application to target localization and beamforming

Abstract The proliferation of wireless sensor networks and the variety of envisioned applications associated with them has motivated the development of distributed algorithms for collaborative processing over networked systems. One of the applications that has attracted the attention of the researchers is that of target localization where the nodes of the network try to estimate the position of an unknown target that lies within its coverage area. Particularly challenging is the problem of estimating the target’s position when we use received signal strength indicator (RSSI) due to the nonlinear relationship between the measured signal and the true position of the target. Many of the existing approaches suffer either from high computational complexity (e.g., particle filters) or lack of accuracy. Further, many of the proposed solutions are centralized which make their application to a sensor network questionable. Depending on the application at hand and, from a practical perspective it could be convenient to find a balance between localization accuracy and complexity. Into this direction we approach the maximum likelihood location estimation problem by solving a suboptimal (and more tractable) problem. One of the main advantages of the proposed scheme is that it allows for a decentralized implementation using distributed processing tools (e.g., consensus and convex optimization) and therefore, it is very suitable to be implemented in real sensor networks. If further accuracy is needed an additional refinement step could be performed around the found solution. Under the assumption of independent noise among the nodes such local search can be done in a fully distributed way using a distributed version of the Gauss-Newton method based on consensus. Regardless of the underlying application or function of the sensor network it is al¬ways necessary to have a mechanism for data reporting. While some approaches use a special kind of nodes (called sink nodes) for data harvesting and forwarding to the outside world, there are however some scenarios where such an approach is impractical or even impossible to deploy. Further, such sink nodes become a bottleneck in terms of traffic flow and power consumption. To overcome these issues instead of using sink nodes for data reporting one could use collaborative beamforming techniques to forward directly the generated data to a base station or gateway to the outside world. In a dis-tributed environment like a sensor network nodes cooperate in order to form a virtual antenna array that can exploit the benefits of multi-antenna communications. In col-laborative beamforming nodes synchronize their phases in order to add constructively at the receiver. Some of the inconveniences associated with collaborative beamforming techniques is that there is no control over the radiation pattern since it is treated as a random quantity. This may cause interference to other coexisting systems and fast bat-tery depletion at the nodes. Since energy-efficiency is a major design issue we consider the development of a distributed collaborative beamforming scheme that maximizes the network lifetime while meeting some quality of service (QoS) requirement at the re¬ceiver side. Using local information about battery status and channel conditions we find distributed algorithms that converge to the optimal centralized beamformer. While in the first part we consider only battery depletion due to communications beamforming, we extend the model to account for more realistic scenarios by the introduction of an additional random energy consumption. It is shown how the new problem generalizes the original one and under which conditions it is easily solvable. By formulating the problem under the energy-efficiency perspective the network’s lifetime is significantly improved. Resumen La proliferación de las redes inalámbricas de sensores junto con la gran variedad de posi¬bles aplicaciones relacionadas, han motivado el desarrollo de herramientas y algoritmos necesarios para el procesado cooperativo en sistemas distribuidos. Una de las aplicaciones que suscitado mayor interés entre la comunidad científica es la de localization, donde el conjunto de nodos de la red intenta estimar la posición de un blanco localizado dentro de su área de cobertura. El problema de la localization es especialmente desafiante cuando se usan niveles de energía de la seal recibida (RSSI por sus siglas en inglés) como medida para la localization. El principal inconveniente reside en el hecho que el nivel de señal recibida no sigue una relación lineal con la posición del blanco. Muchas de las soluciones actuales al problema de localization usando RSSI se basan en complejos esquemas centralizados como filtros de partículas, mientas que en otras se basan en esquemas mucho más simples pero con menor precisión. Además, en muchos casos las estrategias son centralizadas lo que resulta poco prácticos para su implementación en redes de sensores. Desde un punto de vista práctico y de implementation, es conveniente, para ciertos escenarios y aplicaciones, el desarrollo de alternativas que ofrezcan un compromiso entre complejidad y precisión. En esta línea, en lugar de abordar directamente el problema de la estimación de la posición del blanco bajo el criterio de máxima verosimilitud, proponemos usar una formulación subóptima del problema más manejable analíticamente y que ofrece la ventaja de permitir en¬contrar la solución al problema de localization de una forma totalmente distribuida, convirtiéndola así en una solución atractiva dentro del contexto de redes inalámbricas de sensores. Para ello, se usan herramientas de procesado distribuido como los algorit¬mos de consenso y de optimización convexa en sistemas distribuidos. Para aplicaciones donde se requiera de un mayor grado de precisión se propone una estrategia que con¬siste en la optimización local de la función de verosimilitud entorno a la estimación inicialmente obtenida. Esta optimización se puede realizar de forma descentralizada usando una versión basada en consenso del método de Gauss-Newton siempre y cuando asumamos independencia de los ruidos de medida en los diferentes nodos. Independientemente de la aplicación subyacente de la red de sensores, es necesario tener un mecanismo que permita recopilar los datos provenientes de la red de sensores. Una forma de hacerlo es mediante el uso de uno o varios nodos especiales, llamados nodos “sumidero”, (sink en inglés) que actúen como centros recolectores de información y que estarán equipados con hardware adicional que les permita la interacción con el exterior de la red. La principal desventaja de esta estrategia es que dichos nodos se convierten en cuellos de botella en cuanto a tráfico y capacidad de cálculo. Como alter¬nativa se pueden usar técnicas cooperativas de conformación de haz (beamforming en inglés) de manera que el conjunto de la red puede verse como un único sistema virtual de múltiples antenas y, por tanto, que exploten los beneficios que ofrecen las comu¬nicaciones con múltiples antenas. Para ello, los distintos nodos de la red sincronizan sus transmisiones de manera que se produce una interferencia constructiva en el recep¬tor. No obstante, las actuales técnicas se basan en resultados promedios y asintóticos, cuando el número de nodos es muy grande. Para una configuración específica se pierde el control sobre el diagrama de radiación causando posibles interferencias sobre sis¬temas coexistentes o gastando más potencia de la requerida. La eficiencia energética es una cuestión capital en las redes inalámbricas de sensores ya que los nodos están equipados con baterías. Es por tanto muy importante preservar la batería evitando cambios innecesarios y el consecuente aumento de costes. Bajo estas consideraciones, se propone un esquema de conformación de haz que maximice el tiempo de vida útil de la red, entendiendo como tal el máximo tiempo que la red puede estar operativa garantizando unos requisitos de calidad de servicio (QoS por sus siglas en inglés) que permitan una decodificación fiable de la señal recibida en la estación base. Se proponen además algoritmos distribuidos que convergen a la solución centralizada. Inicialmente se considera que la única causa de consumo energético se debe a las comunicaciones con la estación base. Este modelo de consumo energético es modificado para tener en cuenta otras formas de consumo de energía derivadas de procesos inherentes al funcionamiento de la red como la adquisición y procesado de datos, las comunicaciones locales entre nodos, etc. Dicho consumo adicional de energía se modela como una variable aleatoria en cada nodo. Se cambia por tanto, a un escenario probabilístico que generaliza el caso determinista y se proporcionan condiciones bajo las cuales el problema se puede resolver de forma eficiente. Se demuestra que el tiempo de vida de la red mejora de forma significativa usando el criterio propuesto de eficiencia energética.

Damage localization and failure locus under biaxial loading in glass-fiber nonwoven felts

The pattern of damage localization and fracture under uniaxial and biaxial tension was studied in glass–fiber nonwoven felts. The analyses were carried out within the framework of the finite-element simulation of plain and notched specimens in which the microstructure of the felt, made up of fiber bundles connected at the cross point through an organic binder, was explicitly represented. Following previous experimental observations, fracture by interbundle decohesion and energy dissipation by frictional sliding between the bundles were included in the model. It was found that the failure path in these materials was controlled by the maximum applied normal stress, regardless of the loading path, and that the failure locus under biaxial tension was well represented by the von Mises failure criteria. The notch sensitivity of the nonwoven felts was limited and the presence of a notch did not modify the failure path.

Detection and Isolation of Simultaneous Additive and Parametric Faults in Nonlinear Stochastic Dynamical Systems

This paper presents a new fault detection and isolation scheme for dealing with simultaneous additive and parametric faults. The new design integrates a system for additive fault detection based on Castillo and Zufiria, 2009 and a new parametric fault detection and isolation scheme inspired in Munz and Zufiria, 2008 . It is shown that the so far existing schemes do not behave correctly when both additive and parametric faults occur simultaneously; to solve the problem a new integrated scheme is proposed. Computer simulation results are presented to confirm the theoretical studies.