Characterization of SKT1, an Inwardly Rectifying Potassium Channel from Potato, by Heterologous Expression in Insect Cells

A cDNA encoding a novel, inwardly rectifying K+ (K+in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K+in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K+in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than −60 mV. The pharmacological inhibitor Cs+, when applied externally, inhibited SKT1-mediated K+in currents half-maximally with an inhibitor concentration (IC50) of 105 μm. An almost identical high Cs+ sensitivity (IC50 = 90 μm) was found for the potato guard-cell K+in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.

Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

Health care, information needs, and outreach: reaching Ohio's rural citizens

As a rural state, Ohio has a vital interest in addressing rural health and information needs. NetWellness is a Web-based consumer health information service that focuses on the needs of the residents of Ohio. Health sciences faculty from the state's three Carnegie Research I universities—University of Cincinnati, Case Western Reserve University, and The Ohio State University—create and evaluate content and provide Ask an Expert service to all visitors. Through partnerships at the state and local levels, involving public, private, commercial, and noncommercial organizations, NetWellness has grown from a regional demonstration project in 1995 to a key statewide service. Collaboration with public libraries, complemented by alliances with kindergarten through twelfth grade agencies, makes NetWellness Ohio's essential health information resource.

Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.

Plants possess multiple resistance mechanisms that guard against pathogen attack. Among these are inducible systems such as systemic acquired resistance (SAR). SAR is activated by pathogen exposure and leads to an increase in salicylic acid (SA), high-level expression of SAR-related genes, and resistance to a spectrum of pathogens. To identify components of the signal transduction pathways regulating SAR, a mutant screen was developed that uses 2,6-dichloroisonicotinic acid as an activator of SAR gene expression and pathogen resistance, followed by assays for resistance to the fungal pathogen Peronospora parasitica. Mutants from this screen were subsequently examined to assess their defense responses. We describe here a recessive mutation that causes a phenotype of insensitivity to chemical and biological inducers of SAR genes and resistance. These data indicate the existence of a common signaling pathway that couples these diverse stimuli to induction of SAR genes and resistance. Because of its non-inducible immunity phenotype, we call this mutant nim1. Although nim1 plants fail to respond to SA, they retain the ability to accumulate wild-type levels of SA, a probable endogenous signal for SAR. Further, the ability of nim1 plants to support growth of normally incompatible races of a fungal pathogen indicates a role for this pathway in expression of genetically determined resistance, consistent with earlier findings for transgenic plants engineered to break down SA. These results suggest that the wild-type NIM1 gene product functions in a pathway regulating acquired resistance, at a position downstream of SA accumulation and upstream of SAR gene induction and expression of resistance.

Effects of mechanical signaling on plant cell cytosolic calcium.

Mechanical signals are important influences on the development and morphology of higher plants. Using tobacco transformed with the Ca(2+)-sensitive luminescent protein aequorin, we recently reported the effects of mechanical signals of touch and wind on the luminescence and thus intracellular calcium of young seedlings. When mesophyll protoplasts are isolated from these transgenic tobacco plants and mechanically stimulated by swirling them in solution, cytoplasmic Ca2+ increases immediately and transiently up to 10 microM, and these transients are unaffected by an excess of EGTA in the medium. The size of the transient effect is related to the strength of swirling. Epidermal strips isolated from transgenic tobacco leaves and containing only viable guard cells and trichomes also respond to the strength of swirling in solution and can increase their cytoplasmic Ca2+ transiently up to 10 microM. Finally, the moss Physcomitrella patens containing recombinant aequorin exhibits transient increases in cytoplasmic Ca2+ up to 5 microM when swirled in solution. This effect is strongly inhibited by ruthenium red. Our data indicate that the effect of mechanical stimulation can be found in a number of different cell types and in a lower plant as well as tobacco and suggest that mechanoperception and the resulting increase in cytoplasmic Ca2+ may be widespread.

In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.

Strong natural selection causes microscale allozyme variation in a marine snail.

Natural selection is one of the most fundamental processes in biology. However, there is still a controversy over the importance of selection in microevolution of molecular traits. Despite the general lack of data most authors hold the view that selection on molecular characters may be important, but at lower rates than selection on most phenotypic traits. Here we present evidence that natural selection may contribute substantially to molecular variation on a scale of meters only. In populations of the marine snail Littorina saxatilis living on exposed rocky shores, steep microclines in allele frequencies between splash and surf zone groups are present in the enzyme aspartate aminotransferase (allozyme locus Aat; EC. 2.6.1.1). We followed one population over 7 years, including a period of strong natural perturbation. The surf zone part of the population dominated by the allele Aat100 was suddenly eliminated by a bloom of a toxin-producing microflagellate. Downshore migration of splash zone snails with predominantly Aat120 alleles resulted in a drastic increase in surf zone frequency of Aat120, from 0.4 to 0.8 over 2 years. Over the next four to six generations, however, the frequency of Aat120 returned to the original value. We estimated the coefficient of selection of Aat120 in the surf zone to be about 0.4. Earlier studies show similar or even sharper Aat clines in other countries. Thus, we conclude that microclinal selection is an important evolutionary force in this system.

Pontes protendidas de Eucalipto citriodora

O conceito de pontes de madeira em tabuleiro laminado protendido surgiu na década de 70, no Canadá, como forma de reabilitação para tabuleiros laminados pregados. Diversas pesquisas foram desenvolvidas para verificar o desempenho estrutural e a durabilidade do sistema, e estes estudos comprovaram a eficácia do método. Devido ao enorme déficit de pequenas e médias pontes em todo o seu território, o Brasil tem buscado cada vez mais materiais e tecnologias alternativas economicamente competitivas para sua construção. Partindo de pesquisas desenvolvidas em outros países, desde 1993 começaram a ser desenvolvidas pesquisas nacionais para verificar a viabilidade em se utilizar madeiras brasileiras para construção de pontes, e a resposta, mais uma vez, foi positiva. Este trabalho tem como objetivos o estudo teórico e experimental das pontes de eucalipto protendidas transversalmente, através do projeto e construção da primeira ponte protendida de madeira da América do Sul. Por meio de provas de carga, foi avaliado o desempenho da ponte e a influência dos guarda-rodas e defensas na rigidez do tabuleiro. Os resultados mostram que o sistema protendido de eucalipto é uma ótima alternativa para o Brasil.

Back in Blue: The Successful Reintegration of Combat Veteran Law Enforcement Officers.

In the past ten plus years, several million national guard and reserve component military personnel have been deployed in support of the global war on terrorism. Tens of thousands of those personnel also serve as full-time law enforcement officers in police and sheriff's offices around the country. Life as a law enforcement officer is tough enough, but when combined with the psychological baggage brought on by months of war, reintegrating into civilian life and the role of a law enforcement officer can be extremely difficult. This article discusses a reintegration program specifically for law enforcement agencies that is designed to promote long-term psychological and social health in combat veteran officers. The program's costs are offset by the many assets (leadership, tactical training, etc.) these men and women bring to the department. By committing to the long-term successful reintegration of these individuals, departments enhance their own forces and improve community safety.

Comparison of Couples' Normal Life Cycle Development and Intimacy Distance Regulation

When conceptualizing healthy couple relationships, it is tempting to use a simple framework as a panacea. Unfortunately, this desire for simplicity can lead to a narrow and naive perspective. Individuals interact and are influenced by a variety of factors (i.e., various social systems, multiple context memberships, complex interconnecting exchanges, etc.); consequently, it is necessary to guard against an overly narrow interpretation when examining healthy couple interactions. It is the purpose of this paper to develop one aspect of a complex perspective for healthy couple relationships by comparing couple life cycle development with couple intimacy-distance regulation.

Las relaciones públicas en el tratamiento que los medios de comunicación otorgan a los mensajes de la Guardia Civil de Alicante

El presente trabajo expone las diferencias más significativas en el tratamiento que los medios de comunicación otorgan a las noticias de la Guardia Civil de Alicante en dos períodos diferentes y con modelos conductuales de relaciones públicas muy distintos. En el primero de ellos, la oficina de comunicación de la Institución se dedicaba exclusivamente a remitir notas de prensa a los medios; en el segundo, la conducta comunicativa de la Benemérita se basa en modelos bidireccionales de las relaciones públicas. Los resultados presentados ponen de manifiesto la eficacia limitada del modelo unidireccional utilizado por la OPC con los MMCC, sin embargo, esa misma eficacia comunicativa se manifiesta en su máximo exponente al combinar ambos. La incorporación simultánea de los modelos agente de prensa y simétrico bidireccional (Grunig y Hunt, 2003,73) contribuyen al buen tratamiento otorgado por los medios a la Guardia Civil de Alicante.

Características cineantropométricas en jugadores de baloncesto adolescentes

Introducción: El baloncesto presenta una gran heterogeneidad antropométrica entre jugadores en relación a la posición de juego. El base suele ser el más bajo del equipo pero también el más rápido. El escolta suele ser similar al base, más bajo, rápido y ágil que el resto. El alero es de una altura intermedia entre los jugadores interiores y exteriores, combina la altura con la velocidad, y el pívot tiene un rol más físico, es el más alto y con mayor masa muscular. El objetivo del estudio es describir el perfil cineantropométrico del equipo cadete masculino del Valencia Basket Club. Material y Métodos: Se han evaluado 20 jugadores de baloncesto cadete masculino (15,35±0,59años). Un antropometrista nivel I tomó las medidas según la metodología de la Society for the Advancement of Kinanthropometry (ISAK). Se calculó la composición corporal según el consenso de cineantropometría del Grupo Español de Cineantropometría (GREC), el somatotipo según Heath-Carter, e índices adiposo-musculares. Resultados: Se observaron diferencias en los valores de peso, talla, pliegues cutáneos y composición corporal, de las variables estudiadas de la muestra total y por posición de juego, siendo los valores menores los de la posición base y mayores en los pívots. Se estableció una tendencia creciente de estos parámetros en el siguiente orden: base, escolta, alero y pívot. Conclusiones: El somatotipo global del jugador de baloncesto cadete del Valencia Basket Club es ectomorfo balanceado. Conocer el perfil cineantropométrico puede beneficiar la elaboración de planes de entrenamiento y nutrición, así como reflejar la evolución y dirigirla hacia las características ligadas al rendimiento óptimo.

Letter from David Phips to Colonel Jonathan Snelling regarding escort of Governor Hutchinson to Harvard Commencement, 1773 July 12

David Phips wrote this letter to Colonel Jonathan Snelling from Cambridge on July 12, 1773, to inform him that Massachusetts Governor Thomas Hutchinson had requested the accompaniment of guards during his travels from Milton to Cambridge on July 21, 1773, to attend the Harvard College Commencement exercises. In the letter, Phips informs Snelling that he has issued warrants to the guards, instructing them to congregate at the Sign of the Grey Hound in Roxbury, Massachusetts at eight o'clock on the morning of the 21st. He explains that twelve other men will march, under the command of Sub-Brigadier Sumner, to the Governor's home in Milton to escort him to Roxbury, where the larger party will assemble. These heightened security measures were certainly prompted by political unrest, although this is not stated explicitly in the letter. Phips concludes by saying: "I shall order a dinner for us at Bradish's, where I hope to have the pleasure to dine with you."

Chester Castle, Chester, England, ca. 1745 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: A plan of the castle of Chester : with additional new works errected by order of ... the Earl of Cholmondeley, whereunto is added a project of four bastions in order to defend the antient walls against a regular seige ... by ... Alexander de Lavaux. R. Parr sculp. It was published by Alexander de Lavaux ca. 1745. Scale [ca. 1:300]. The image inside the map neatline is georeferenced to the surface of the earth and fit to the 'British National Grid' coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows fortification features such as buildings, stables, guard houses, towers, quarters, stairs, gates, parade grounds, arsenals, wells, ground cover, and more. Includes also index.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Satgûr:jo:salûko : manuscript, 1890?

Local paper.