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.

Grid-based histogram arithmetic for the probabilistic analysis of functions

The selection of predefined analytic grids (partitions of the numeric ranges) to represent input and output functions as histograms has been proposed as a mechanism of approximation in order to control the tradeoff between accuracy and computation times in several áreas ranging from simulation to constraint solving. In particular, the application of interval methods for probabilistic function characterization has been shown to have advantages over other methods based on the simulation of random samples. However, standard interval arithmetic has always been used for the computation steps. In this paper, we introduce an alternative approximate arithmetic aimed at controlling the cost of the interval operations. Its distinctive feature is that grids are taken into account by the operators. We apply the technique in the context of probability density functions in order to improve the accuracy of the probability estimates. Results show that this approach has advantages over existing approaches in some particular situations, although computation times tend to increase significantly when analyzing large functions.

Transient acoustic response in car cabins with localization of reflections

Due to its small size and the restrictions on source and listener positions, the design of sound reproduction systems for car cabins is particularly cumbersome. In the present project the measurement of the impulse response between a single loudspeaker and a listener position, with special emphasis on the directional characteristics, will be examined. The propagation paths inside a car are very short, meaning that it is very difficult for the existing commercial measurement systems to resolve the different reflections arriving to the listener. This paper propose a first approach of an algorithm based on time difference of arrival along a measurement technique aiming at finding the reflections and their direction of arrival to the listener. To this end a circular microphone array at a known position is employed, along with Maximum-Length Sequences (MLS) measurement technique. The results are processed so as to extract the directional properties, demonstrate the physical limitations that can influence or prevent this detection in practice. Measurements were carried out in a free-field environment (anechoic chamber) making use of different panels closer around the microphone array. RESUMEN. El diseño de sistemas de reproducción de audio para cabinas de coche es especialmente complicado debido al reducido tamaño del espacio y las restricciones de los altavoces y posiciones de escucha de los ocupantes. En el presente proyecto, se examinan mediciones de la respuesta al impulso entre un altavoz y una posición de escucha con especial énfasis en las características direccionales. Los caminos de propagación de las ondas sonoras dentro de un coche son muy cortos, lo que hace difícil para los instrumentos de medida existentes en el mercado determinar las direcciones de llegada de las diferentes reflexiones que llegan a una posición de escucha. Este trabajo propone una primera aproximación de un algoritmo, basado en las diferencias temporales de llegada de una onda a diferentes puntos de medida, y una particular técnica de medida de la respuesta al impulso para obtener las direcciones de llegada de reflexiones a una posición de escucha. Para ello, se emplea una matriz circular de micrófonos en una posición conocida junto con la técnica de medida MLS (Maximum Length Sequence). Los resultados obtenidos son procesados para extraer la dirección de llegada de las reflexiones acústicas y encontrar las limitaciones que influyan en la detección de dichas reflexiones. Las mediciones se llevan a cabo en un entorno de campo libre y utilizando diferentes superficies reflectantes alrededor de la matriz de micrófonos.

Meromorphic continuation of functions and arbitrary distribution of interpolation points

We characterize the region of meromorphic continuation of an analytic function ff in terms of the geometric rate of convergence on a compact set of sequences of multi-point rational interpolants of ff. The rational approximants have a bounded number of poles and the distribution of interpolation points is arbitrary.

Cattle-powered nodes experience in a heterogeneous network for localization of herds

A heterogeneous network, mainly based on nodes that use harvested energy to self-energize is presented and its use demonstrated. The network, mostly kinetically powered, has been used for the localization of herds in grazing areas under extreme climate conditions. The network consists of secondary and primary nodes. The former, powered by a kinetic generator, take advantage of animal movements to broadcast a unique identifier. The latter are battery-powered and gather secondarynode transmitted information to provide it, along with position and time data, to a final base station in charge of the animal monitoring. Because a limited human interaction is desirable, the aim of this network is to reduce the battery count of the system.

On the localization of the personalized PageRank of complex networks

In this paper new results on personalized PageRank are shown. We consider directed graphs that may contain dangling nodes. The main result presented gives an analytical characterization of all the possible values of the personalized PageRank for any node.We use this result to give a theoretical justification of a recent model that uses the personalized PageRank to classify users of Social Networks Sites. We introduce new concepts concerning competitivity and leadership in complex networks. We also present some theoretical techniques to locate leaders and competitors which are valid for any personalization vector and by using only information related to the adjacency matrix of the graph and the distribution of its dangling nodes.

Multiple feature points representation in target localization of wireless visual sensor networks

This paper discusses the target localization problem in wireless visual sensor networks. Additive noises and measurement errors will affect the accuracy of target localization when the visual nodes are equipped with low-resolution cameras. In the goal of improving the accuracy of target localization without prior knowledge of the target, each node extracts multiple feature points from images to represent the target at the sensor node level. A statistical method is presented to match the most correlated feature point pair for merging the position information of different sensor nodes at the base station. Besides, in the case that more than one target exists in the field of interest, a scheme for locating multiple targets is provided. Simulation results show that, our proposed method has desirable performance in improving the accuracy of locating single target or multiple targets. Results also show that the proposed method has a better trade-off between camera node usage and localization accuracy.

Bipolar localization of Bacillus subtilis topoisomerase IV, an enzyme required for chromosome segregation

In Bacillus subtilis, parE and parC were shown to be essential genes for the segregation of replicated chromosomes. Disruption of either one of these genes resulted in failure of the nucleoid to segregate. Purified ParE and ParC proteins reconstituted to form topoisomerase IV (topo IV), which was highly proficient for ATP-dependent superhelical DNA relaxation and decatenation of interlocked DNA networks. By immunofluorescence microscopy and by directly visualizing fluorescence by using green fluorescence protein fusions, we determined that ParC is localized at the poles of the bacteria in rapidly growing cultures. The bipolar localization of ParC required functional ParE, suggesting that topo IV activity is required for the localization. ParE was found to be distributed uniformly throughout the cell. On the other hand, fluorescence microscopy showed that the GyrA and GyrB subunits of gyrase were associated with the nucleoid. Our results provide a physiologic distinction between DNA gyrase and topo IV. The subcellular localization of topo IV provides physical evidence that it may be part of the bacterial segregation machinery.

Nucleolar localization of the Werner syndrome protein in human cells

Werner Syndrome (WS) is a human genetic disorder with many features of premature aging. The gene defective in WS (WRN) has been cloned and encodes a protein homologous to several helicases, including Escherichia coli RecQ, the human Bloom syndrome protein (BLM), and Saccharomyces cerevisiae Sgs1p. To better define the function of WRN protein we have determined its subcellular localization. Indirect immunofluorescence using polyclonal anti-human WRN shows a predominant nucleolar localization. Studies of WRN mutant cells lines confirmed the specificity of antibody recognition. No difference was seen in the subcellular localization of the WRN protein in a variety of normal and transformed human cell lines, including both carcinomas and sarcomas. The nucleolar localization of human WRN protein was supported by the finding that upon biochemical subcellular fractionation, WRN protein is present in an increased concentration in a subnuclear fraction enriched for nucleolar proteins. We have also determined the subcellular localization of the mouse WRN homologue (mWRN). In contrast to human WRN protein, mWRN protein is present diffusely throughout the nucleus. Understanding the function of WRN in these organisms of vastly differing lifespan may yield new insights into the mechanisms of lifespan determination.

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30–42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

Aberrant intracellular localization of Varicella-Zoster virus regulatory proteins during latency

Varicella-Zoster virus (VZV) is a herpesvirus that becomes latent in sensory neurons after primary infection (chickenpox) and subsequently may reactivate to cause zoster. The mechanism by which this virus maintains latency, and the factors involved, are poorly understood. Here we demonstrate, by immunohistochemical analysis of ganglia obtained at autopsy from seropositive patients without clinical symptoms of VZV infection that viral regulatory proteins are present in latently infected neurons. These proteins, which localize to the nucleus of cells during lytic infection, predominantly are detected in the cytoplasm of latently infected neurons. The restriction of regulatory proteins from the nucleus of latently infected neurons might interrupt the cascade of virus gene expression that leads to a productive infection. Our findings raise the possibility that VZV has developed a novel mechanism for maintenance of latency that contrasts with the transcriptional repression that is associated with latency of herpes simplex virus, the prototypic alpha herpesvirus.

Mitochondrial localization of a NADR-dependent isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5′ region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.

Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei

In Trypanosoma brucei, transcription by RNA polymerase II and 5′ capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I. In a single trypanosome, only one VSG expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using fluorescence in situ hybridization. With probes that cover about 50 kb of the active 221 expression site, we detected nuclear transcripts of this site in a single fluorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by fluorescent DNA in situ hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active VSG expression site does not take place in the nucleolus.

Rer1p as common machinery for the endoplasmic reticulum localization of membrane proteins

Rer1p, a Golgi membrane protein, is required for the correct localization of an endoplasmic reticulum (ER) membrane protein, Sec12p, by a retrieval mechanism from the cis-Golgi to the ER. To test whether or not the role of Rer1p is common to multiple ER membrane proteins, we examined the localization of two other ER membrane proteins, Sec71p and Sec63p, in the wild-type and rer1 mutant yeast cells, using their fusions with an α-mating factor precursor (Mfα1p). Although Sec71p and Sec63p have completely different topology from Sec12p, their Mfα1p fusion proteins were also mislocalized to the trans-Golgi in the rer1 mutant. Overexpression of these fusions caused their mislocalization to the trans-Golgi even in the wild-type cells, and this mislocalization was partially suppressed by the co-overexpression of Rer1p. Either Sec71p or an artificial chimeric protein whose ER localization depends on Rer1p gave a competitive effect on the localization of the Mfα1-Sec71p fusion, which was abolished in rer1. Thus, Rer1p appears to be one of the common limiting components in the retrieval machinery for ER membrane proteins. The results also suggest that Sec71p and Sec63p depend on ER-Golgi recycling, at least partly, for ER localization. We also examined the effect of a mutation in α-COP, a subunit of yeast coatomer, on the localization of these ER membrane proteins. The Mfα1p fusions of Sec12p, Sec71p, and Sec63p were all more or less mislocalized in ret1–1. These observations imply that the roles of Rer1p and coatomer are much more general than thought before.