bluuebunny/crossref_metadata_2025 huggingface dataset:
A Polytene Chromosome Analysis of the
Anopheles gambiae
Species Complex
Authors: Coluzzi Mario , Sabatini Adriana , della Torre Alessandra , Di Deco Maria Angela , Petrarca Vincenzo
Published: November 2002

Field-collected specimens of all known taxa in the
Anopheles gambiae
complex were analyzed on the basis of chromosome inversions with reference to a standard polytene chromosome map. The phylogenetic relationships among the seven described species in the complex could be inferred from the distribution of fixed inversions. Nonrandom patterns of inversion distribution were observed and, particularly on chromosome arm 2R, provided evidence for genetically distinct populations in
A. gambiae
,
A. arabiensis
, and
A. melas
. In
A. gambiae
from Mali, stable genetic differentiation was observed even in populations living in the same region, suggesting a process of incipient speciation which is being confirmed by studies with molecular markers. The possible role of chromosome differentiation in speciation of the
A. gambiae
complex and in the emergence of distinct chromosomal forms within the nominal species is discussed in relation to human malaria.
Elimination of Channel-Forming Activity by Insertional Inactivation of thep13Gene inBorrelia burgdorferi
Authors: Östberg Yngve , Pinne Marija , Benz Roland , Rosa Patricia , Bergström Sven
Published: November 2002

P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent,Borrelia burgdorferi. The aim of this study was to investigate the function of the P13 protein. Here, we inactivated thep13gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in thep13mutant compared to wild-typeB. burgdorferi, indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in thep13mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivatedp13gene could be detected under normal growth conditions. The inactivation ofp13is the first reported inactivation of a gene encoding an integral outer membrane protein inB. burgdorferi. Here, we describe both genetic and biophysical experiments indicating that P13 inB. burgdorferiis an outer membrane protein with porin activity.
Different Spectra of Stationary-Phase Mutations in Early-Arising versus Late-Arising Mutants of
Pseudomonas putida
: Involvement of the DNA Repair Enzyme MutY and the Stationary-Phase Sigma Factor RpoS
Authors: Saumaa Signe , Tover Andres , Kasak Lagle , Kivisaar Maia
Published: November 2002

Stationary-phase mutations occur in populations of stressed, nongrowing, and slowly growing cells and allow mutant bacteria to overcome growth barriers. Mutational processes in starving cells are different from those occurring in growing bacteria. Here, we present evidence that changes in mutational processes also take place during starvation of bacteria. Our test system for selection of mutants based on creation of functional promoters for the transcriptional activation of the phenol degradation genes
pheBA
in starving
Pseudomonas putida
enables us to study base substitutions (C-to-A or G-to-T transversions), deletions, and insertions. We observed changes in the spectrum of promoter-creating mutations during prolonged starvation of
Pseudomonas putida
on phenol minimal plates. One particular C-to-A transversion was the prevailing mutation in starving cells. However, with increasing time of starvation, the importance of this mutation decreased but the percentage of other types of mutations, such as 2- to 3-bp deletions, increased. The rate of transversions was markedly elevated in the
P. putida
MutY-defective strain. The occurrence of 2- to 3-bp deletions required the stationary-phase sigma factor RpoS, which indicates that some mutagenic pathway is positively controlled by RpoS in
P. putida
.
Analysis of Infection Thread Development Using Gfp- and DsRed-Expressing
Sinorhizobium meliloti
Authors: Gage Daniel J.
Published: November 2002

Sinorhizobium meliloti
growth inside infection threads was monitored after inoculation of alfalfa with red- or green-tagged bacteria. Most threads were populated with single bacterial types. Mixed infections were present but gave mixed nodules less often than expected. These patterns are explained by a model describing bacterial growth during infection.
POWER ESTIMATOR DEVELOPMENT FOR EMBEDDED SYSTEM MEMORY TUNING
Authors: VAHID FRANK , GIVARGIS TONY , COTTERELL SUSAN
Published: November 2002

Memory accesses account for a large percentage of total power in microprocessor-based embedded systems. The increasing use of microprocessor cores and synthesis, rather than prefabricated microprocessor chips, creates the opportunity to tune a memory hierarchy to the one program that will execute in the embedded system. Such tuning requires fast and accurate estimation of the power and performance of different memory configurations. We describe a general three-step approach to developing such estimators, based on our experiences on several different projects. Each step is increasingly fast, using the previous step to gauge accuracy. The first step uses high-level functional simulation, the second step uses trace simulation, and the third step uses equations. A tool developer can follow these three steps to create a powerful environment for core users to support synthesis of the best memory hierarchy for a particular embedded system. The approach can be applied to components other than memory also.
CODE COVERAGE-BASED POWER ESTIMATION TECHNIQUES FOR MICROPROCESSORS
Authors: QU GANG , KAWABE NAOYUKI , USAMI KIMIYOSHI , POTKONJAK MIODRAG
Published: November 2002

We have developed a function-level power estimation methodology for predicting the power dissipation of embedded software. For a given microprocessor core, we empirically build the "power data bank", which stores the power information of the built-in library functions and basic instructions. To estimate the average power of an embedded software on this core, we first decompose the machine code into library functions and user-defined functions. We then use program profiling/tracing tools to get the execution information of the target software. Next, we evaluate the total energy consumption and execution time based on the "power data bank", and their ratio is taken as the average power. High efficiency is achieved because no power simulator is used once the "power data bank" is built. We apply this method to a commercial microprocessor core and get power estimates with an average error of 3%. Using this method, microprocessor vendors can provide users the "power data bank" without releasing details of the core to help users get early power estimates and eventually guide power optimization.
ON THE 1/f NOISE IN 0.15 μm FULLY DEPLETED SOI/MOS TRANSISTORS
Authors: HAENDLER S. , JOMAAH J. , BALESTRA F.
Published: November 2002

The low frequency noise (LFN) behavior in 0.15 μm N-channel fully depleted SOI/MOS transistors is investigated, both in ohmic and saturation regimes. The extraction of the interface trap density is also carried out. Moreover, the existence of a moderate kink effect and its influence on LF noise in fully depleted devices are shown.
Dissociation of Regional Adaptations to Ischemia and Global Myolysis in an Accelerated Swine Model of Chronic Hibernating Myocardium
Authors: Thomas Salome A. , Fallavollita James A. , Suzuki Gen , Borgers Marcel , Canty John M.
Published: November 2002

We tested the hypothesis that an acute critical limitation in coronary flow reserve could rapidly recapitulate the physiological, molecular, and morphological phenotype of hibernating myocardium. Chronically instrumented swine were subjected to a partial occlusion to produce acute stunning, followed by reperfusion through a critical stenosis. Stenosis severity was adjusted serially so that hyperemic flow was severely reduced yet always higher than the preocclusion resting level. After 24 hours, resting left anterior descending coronary artery (LAD) wall thickening had decreased from 36.3±4.0% to 25.5±3.7% (
P
<0.05), whereas resting flow had remained normal (67±6 versus 67±8 mL/min, respectively). Although peak hyperemic flow exceeded the prestenotic value, resting flow (45±10 mL/min) and LAD wall thickening (17.0±5.0%) progressively decreased after 2 weeks, when physiological features of hibernating myocardium had developed. Regional reductions in sarcoplasmic reticulum proteins were present in hibernating myocardium but absent in stunned myocardium evaluated after 24 hours. Histological analysis showed an increase in connective tissue along with myolysis (myofibrillar loss per myocyte >10%) and increased glycogen typical of hibernating myocardium in the LAD region (33±3% of myocytes from animals with hibernating myocardium versus 15±4% of myocytes from sham-instrumented animals,
P
<0.05). Surprisingly, the frequency of myolysis was similar in normally perfused remote regions from animals with hibernating myocardium (32±7%). We conclude that the regional physiological and molecular characteristics of hibernating myocardium develop rapidly after a critical limitation in flow reserve. In contrast, the global nature of myolysis and increased glycogen content dissociate them from the intrinsic adaptations to ischemia. These may be related to chronic elevations in preload but appear unlikely to contribute to chronic contractile dysfunction.
Involvement of Src Family Protein Tyrosine Kinases in Ca
2+
Sensitization of Coronary Artery Contraction Mediated by a Sphingosylphosphorylcholine-Rho-Kinase Pathway
Authors: Nakao Fumiaki , Kobayashi Sei , Mogami Kimiko , Mizukami Yoichi , Shirao Satoshi , Miwa Saori , Todoroki-Ikeda Natsuko , Ito Masaaki , Matsuzaki Masunori
Published: November 2002

We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase–mediated Ca
2+
sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca
2+
sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca
2+
]
i
levels and the Ca
2+
-dependent contraction induced by high K
+
depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase–dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca
2+
sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.
Long-Term Reward Prediction in TD Models of the Dopamine System
Authors: Daw Nathaniel D. , Touretzky David S.
Published: November 2002

This article addresses the relationship between long-term reward predictions and slow-timescale neural activity in temporal difference (TD) models of the dopamine system. Such models attempt to explain how the activity of dopamine (DA) neurons relates to errors in the prediction of future rewards. Previous models have been mostly restricted to short-term predictions of rewards expected during a single, somewhat artificially defined trial. Also, the models focused exclusively on the phasic pause-and-burst activity of primate DA neurons; the neurons' slower, tonic background activity was assumed to be constant. This has led to difficulty in explaining the results of neurochemical experiments that measure indications of DA release on a slow timescale, results that seem at first glance inconsistent with a reward prediction model. In this article, we investigate a TD model of DA activity modified so as to enable it to make longer-term predictions about rewards expected far in the future. We show that these predictions manifest themselves as slow changes in the baseline error signal, which we associate with tonic DA activity. Using this model, we make new predictions about the behavior of the DA system in a number of experimental situations. Some of these predictions suggest new computational explanations for previously puzzling data, such as indications from microdialysis studies of elevated DA activity triggered by aversive events.
Two Timescale Analysis of the Alopex Algorithm for Optimization
Authors: Sastry P. S. , Magesh M. , Unnikrishnan K. P.
Published: November 2002

Alopex is a correlation-based gradient-free optimization technique useful in many learning problems. However, there are no analytical results on the asymptotic behavior of this algorithm. This article presents a new version of Alopex that can be analyzed using techniques of two timescale stochastic approximation method. It is shown that the algorithm asymptotically behaves like a gradient-descent method, though it does not need (or estimate) any gradient information. It is also shown, through simulations, that the algorithm is quite effective.
Estudio de impacto ambiental de la Plaza Berengé de Granollers: aproximación cualitativa
Authors: Aparicio Simón , Cátedra Toni , Jardí Anna , Vivas Pep
Published: November 2002

En este estudio pretendemos realizar la evaluación del impacto ambiental de una plaza de Granollers (Barcelona). El interés está motivado por los artículos aparecidos en una revista local que evidenciaban una actitud negativa de la mayoría de la población hacia esta plaza. El objeto de análisis que será prioritario será el lenguaje obtenido a partir de entrevistas semi-estructuradas. ¿Por qué desde la Psicología Ambiental? Principalmente por dos razones. En primer lugar, por la necesidad de la comunicación y coordinación de todos/as aquellos/as que están implicados en la planificación de un medio ambiente construido y en que éste cumpla la función y uso deseado. En segundo lugar, para elaborar un diagnóstico ad hoc y a priori de los espacios construidos. En definitiva se pretende aportar un marco de referencia para que los/las políticos/as, diseñadores/as, arquitectos/as y urbanistas escuchen a los/las psicólogos/as ambientales, antes de proyectar y que escuchen a aquellos/as que utilizan lo proyectado.
Estructura de las actitudes ambientales: ¿orientación general o especialización actitudinal?
Authors: Corraliza José A. , Berenguer Jaime
Published: November 2002

El estudio de las actitudes ambientales se articula sobre centros de interés diferenciados. Algunos trabajos están orientados por la definición de la estructura factorial sobre la que se organizan las actitudes ambientales. Otros ponen el acento en la investigación de los procedimientos más eficaces para el cambio y modificación de actitudes ambientales. Los resultados de investigación muestran un panorama de resultados escasamente acumulativos, cuya relevancia depende de las muestras de participantes utilizadas o de los temas ambientales objeto de estudio. Este trabajo pretende establecer, en base a las respuestas a un cuestionario de actitudes ambientales de una muestra de 396 personas, la pertinencia de la diferenciación entre actitudes ambientales consideradas como un conjunto de creencias y/o juicios que reflejan una valoración general de la problemática ambiental frente a creencias y/o juicios focalizados sobre acciones ambientales en función de la estimación subjetiva de los costes. Los resultados confirman, de un lado, que las personas pueden adoptar una estructura actitudinal de orientación general de un signo, y actitudes especializadas de otro signo y que, por otro lado, se produce una diferente valoración de la problemática ambiental en un nivel general o en función de la exigencia que la conducta tenga para el sujeto (eficiencia, sacrificio, derroche).
Evaluación de las actitudes proambientales en trabajadores de grandes organizaciones
Authors: García-Mira Ricardo , Santos Ma Carmen , Gómez-Durán Bernardo , Romay José , Fernández Ma Angeles
Published: November 2002

Este trabajo se enmarca dentro de un proyecto de investigación que pretende el diseño de un programa de intervención, a través del que se trata de dilucidar el papel de las grandes organizaciones en la integración de actitudes ambientales sostenibles en la vida de cada día.

El planteamiento de la investigación surge del reconocimiento de la necesidad de fomentar el cambio en las prácticas y actitudes de las personas hacia el entorno, y en el presente trabajo se informará de los resultados obtenidos en la primera fase, consistentes en la evaluación de las actitudes proambientales y de las dimensiones más relevantes que caracterizan la respuesta ambiental.

Mediante un instrumento de cuestionario, se pretende desglosar cada componente implicado en la actitud, de acuerdo con el modelo de Rosenberg y Hovland (1960) y la obtención de la estructura dimensional que caracteriza la respuesta evaluativa de los trabajadores de dos organizaciones públicas. Las dimensiones obtenidas son analizadas por medio de procedimientos multivariados y comentadas en relación con un conjunto de escalas de preocupación medioambiental.
El significado del trabajo y la innovación tecnológica: aportaciones metodológicas
Authors: Gimeno Miguel Ángel , Salanova Marisa
Published: November 2002

Mediante el presente trabajo se establecen las aportaciones que diferentes aproximaciones metodológicas pueden ofrecer en un campo tan dinámico y flexible como es el del cambio organizacional y actitudinal, más concretamente en la interinfluencia que se puede dar entre las innovaciones tecnológicas y el significado percibido del trabajo. Se ha observado que ambas aproximaciones metodológicas (cuantitativa y cualitativa) muestran resultados y conclusiones similares. Sin embargo, la aproximación cualitativa resulta mucho más enriquecedora cuando se analizan aspectos novedosos, globales y cambiantes, sobre los que no existen teorías previas o específicas.
El cuestionario de valores de Schwartz (CVS): propuesta de adaptación en el formato de respuesta
Authors: Gouveia Valdiney V. , Clemente Miguel , Vidal Miguel Ángel
Published: November 2002

El objetivo de este estudio fue comprobar la posibilidad de simplificar el formato de contestación del Cuestionario de Valores de Schwartz (CVS), en su adaptación española (Ros y Grad, 1991). Una muestra de 176 universitarios ha sido considerada, siendo la mayoría mujeres y solteros; éstos contestaron dos versiones del CVS: la original y la adaptada, y una escala sobre individualismo-colectivismo (Singelis y colsi., 1995). Cuando se comparan las dos versiones en función de los supuestos teóricos, los datos apoyan la adecuación de la versión alternativa. Asimismo son equivalentes respecto de un criterio externo: la religiosidad. Se recomienda el uso de la versión alternativa, aunque se realizan algunas puntualizaciones.
Sobre la definición del conflicto interpersonal: aplicación del cluster analysis al estudio semántico
Authors: Infante Eduardo
Published: November 2002

Siguiendo las iniciativas de varios autores al definir el conflicto y su naturaleza (Pondy, 1967; Fink, 1968; Thomas, 1992), se analizan 78 definicionessobre conflicto interpersonal halladas en diversas fuentes desde 1933 a 1996. El objetivo es el de esclarecer el concepto de conflicto y su naturaleza. Mediante un cluster analysis, se determinaron los tipos de definiciones existentes en la historia de su literatura. Las categorías incluidas fueron las siguientes: (1) fase de conflicto; (2) base del conflicto; (3) percepción; (4) incompatibilidad; (5) vía de interacción; (6) número de implicados; (7) poder (interpersonal); (8) interdependencia; y (9), naturaleza.

Los resultados indican que el conflicto interpersonal es mayoritariamente definido como un proceso-producto de carácter subjetivo-cognitivo que implica percepciones de metas incompatiblespor, al menos, dos individuos. Ninguna otra categoría se mencionó con relevancia. El cluster analysis identificódos grupos de definiciones:positivas (32%) y negativas (68%). La mayoría de las definiciones actuales (posteriores a 1984) se incluyeron más en el segundo grupo. Finalmente, se propone una nueva definición sobre conflicto interpersonal.
Optimismo privado y pesimismo público: un estudio experimental
Authors: Lucas Antonio
Published: November 2002

La satisfacción que la gente dice experimentar hacia cuestiones personales no se complementa con un sentimiento análogo hacia lo público. Los indicadores macrosociales de los últimos 20 años en los países más desarrollados muestran altas cotas de satisfacción vital subjetiva, pero también una creciente insatisfacción hacia cuestiones más generales. Esta manifestación de satisfacción/insatisfacción es investigada en un estudio con un diseño factorial 3 × 2. La primera variable es la personalización de la opinión, pudiendo darse ésta en términos bien colectivos (la gente), bien impersonales (el país), o bien personales (yo). La segunda es tipo de análisis de la sociedad, optimista o pesimista, que lleva a cabo un entrevistado ficticio. Se ha medido el grado de acuerdo con los contenidos de la entrevista y la valoración que se hace del sujeto entrevistado. Los resultados muestran una interacción que va en la línea de las tendencias macrosociales: se empatiza preferentemente con el optimismo personal y el pesimismo social.
Imagen social de las mujeres víctimas de violencia familiar
Authors: Perles Fabiola , Moreno Pilar , Rando Miguel Ángel
Published: November 2002

Entre los estudios que han abordado la problemática de la violencia familiar, algunos se han centrado en el análisis de la percepción social de la víctima de la agresión. El presente trabajo pretende comprobar si las mujeres que son víctimas de agresiones por parte de su pareja son percibidas de forma diferente en función de que se haya producido o no una provocación verbal antes de ser maltratadas. Los sujetos debían responder a una serie de cuestiones sobre un suceso de violencia familiar tras haber leido una noticia de prensa ficticia. En un primer grupo, el suceso de malos tratos no incluía provocación verbal por parte de la mujer; en el segundo y en el tercer grupo el incidente era idéntico, pero incluía dos niveles de provocación, leve y fuerte respectivamente. Entre los resultados más destacados se encontró que el comportamiento de la víctima resulta más compresible cuando se enfrenta verbalmente a su aggressor.
Discriminación étnica en un espacio público. El poder de las normas en el juicio sobre escenas urbanas
Authors: Rebolloso Enrique , Fernández Baltasar , Pozo Carmen
Published: November 2002

El estudio del prejuicio y la discriminación en situaciones públicas y diarias de interacción entre grupos implicados en una cuestión de prejuicio étnico puede ser interesante. Los espacios públicos urbanos tienen niveles variables de estructuración de las interacciones (guiones, normas, reglas del lugar) y podrían relacionarse con la expresión del prejuicio como comportamientos discriminativos. El método de estudio consiste en una simulación de escenarios reales de un pueblo almeriense. Los respondentesdeben anticipar sus recorridos a través de, y sus posiciones de estancia en los lugares. La presencia de inmigrantes y nativos es sistemáticamete variada como situaciones posibles en los lugares (a solas, juntos o separados). Los resultados muestran la existencia de discriminación: los residentes en el área de estudio, así como los respondentes de alto prejuicio, siguen una norma de tipo descriptivo, en función de la posición de otros almerienses en los lugares, cuando los inmigrantes están también presents.
Identidad etnolingüística y construcción de ciudadanía en las Comunidades Autónomas Bilingües (CAB) de España
Authors: Azurmendi María-José , Bourhis Richard Y. , Ros María , García Iñaki
Published: November 2002

En este primer artículo se estudia la identidad etnolingüística resultante de los procesos de identificación con las lenguas y las culturas en contacto en las CAB, obteniendo un continuum identitario en el que los tipos de identidad extremos son: el tipo de identidad Autonómica (Catalana/Gallega/Valenciana/Vasca) y el tipo de identidad Española, que ocuparían los polos del continuum, y el tipo de identidad Autonómico-Española, o bilingüe-bicultural, que se ubicaría en el centro del continuum. Éste ha sido un resultado consistente, obtenido sistemáticamente en todas las CAB. Se han obtenido también otros tipos identitarios, considerados secundarios en este trabajo, tanto por razones teóricas como por razones empíricas, al no darse sistemáticamente en todas las CAB. También se han estudiado las concepciones de ciudadanía que se corresponden con cada grupos según el tipo de identidad, también a cada grupo según el origen geográfico, en relación con las estrategias intergrupales de asimilación y de integración, ya que éstas son las estrategias predominantes de las políticas lingüístico-culturales que concurren en las CAB; también ahora se han obtenido resultados relativamente sistemáticos.
Oral anticoagulation reduces activated protein C less than protein C and other vitamin K–dependent clotting factors
Authors: Simmelink Marleen J. A. , de Groot Philip G. , Derksen Ronald H. W. M. , Fernández José A. , Griffin John H.
Published: November 2002

Oral anticoagulant therapy, which is used for prophylaxis and management of thrombotic disorders, causes similar reductions in plasma levels of vitamin K–dependent procoagulant and anticoagulant clotting factor zymogens. When we measured levels of circulating activated protein C, a physiologically important anticoagulant and anti-inflammatory agent, in patients on oral anticoagulant therapy, the results unexpectedly showed that such therapy decreases levels of activated protein C substantially less than levels of protein C, prothrombin, and factor X, especially at lower levels of prothrombin and factor X. Thus, we suggest that oral anticoagulant therapy results in a relatively increased expression of the protein C pathway compared with procoagulant pathways not only because there is less prothrombin to inhibit activated protein C anticoagulant activity, but also because there is a disproportionately higher level of circulating activated protein C.
Correction of complete interferon-γ receptor 1 deficiency by bone marrow transplantation
Authors: Reuter Ursula , Roesler Joachim , Thiede Christian , Schulz Ansgar , Classen Carl Friedrich , Oelschlägel Uta , Debatin Klaus-Michael , Friedrich Wilhelm
Published: November 2002

Complete interferon-γ receptor 1 (IFNγR1) deficiency is a primary immunodeficiency disease characterized by high susceptibility to recurrent, severe mycobacterial and other intracellular infections. We here report the first successful treatment of the disorder by bone marrow transplantation (BMT). The 8-year-old girl had suffered from recurrent mycobacterial infections in the past and had developed liver cirrhosis with portal hypertension. For conditioning, fractionated total body irradiation (TBI) was used in combination with cyclophosphamide and antithymocyte globulin (ATG). The patient received red cell–depleted bone marrow from her HLA-identical sister. The transplantation course was uneventful and 4 years later, the child remains in excellent clinical condition and free of mycobacterial infections. She has stable mixed lymphohematopoietic chimerism after repeat T-cell transfusions. Liver disease has not further deteriorated. This experience shows that correction of IFNγR1 deficiency is possible by BMT and complications of the disease can be controlled.
Methylation of α-type embryonic globin gene απrepresses transcription in primary erythroid cells
Authors: Singal Rakesh , vanWert Jane M. , Ferdinand Larry
Published: November 2002

The inverse relationship between expression and methylation of β-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian α-type globin genes. The embryonicαπ-globin promoter was unmethylated, andαπ-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the απ promoter associated with loss of expression of απ-globin gene was seen during development in primary erythroid cells. A 315-bpαπ-globin promoter region was cloned in an expression construct (απpGL3E) containing a luciferase reporter gene and SV40 enhancer. The απpGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of απpGL3E plasmid andαπ-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bpαπ-globin gene promoter fragment formed amethyl cytosine-binding proteincomplex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with theαπ-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian α-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.
The death-promoting activity of p53 can be inhibited by distinct signaling pathways
Authors: Lin Yunping , Brown Lauren , Hedley David W. , Barber Dwayne L. , Benchimol Samuel
Published: November 2002

Various cytokines have been shown to protect cells from p53-dependent apoptosis. To investigate the mechanism underlying cytokine-mediated survival, we used a Friend virus–transformed erythroleukemia cell line that expresses a temperature-sensitive p53 allele. These cells express the spleen focus-forming virus-encoded envelope glycoprotein gp55 that allows the cells to proliferate in the absence of erythropoietin (EPO). These cells respond to p53 activation at 32°C by undergoing G1 cell cycle arrest and apoptosis. In the presence of EPO, p53 activation leads only to prolonged but viable G1 arrest. These findings indicate that EPO functions as a survival factor and that gp55/EPO receptor signaling is distinct from EPO/EPO receptor signaling. We demonstrate that p53-dependent apoptosis results in mitochondrial damage as shown by loss of mitochondrial membrane potential, increase in intracellular calcium, and release of mitochondrial cytochrome c into the cytosol. EPO prevented all of these changes including the subsequent activation of caspases. We identify an intrinsic phosphatidylinositol-3′-OH kinase/protein kinase B (PI3′K/PKB)–dependent survival pathway that is constitutively active in these cells. This survival pathway limits p53-dependent apoptosis. We propose that EPO promotes survival through a distinct pathway that is dependent on JAK2 but independent of STAT5 and PI3′K.
Gene therapy of RAG-2−/− mice: sustained correction of the immunodeficiency
Authors: Yates Frank , Malassis-Séris Michèle , Stockholm Daniel , Bouneaud Cécile , Larousserie Frédérique , Noguiez-Hellin Patricia , Danos Olivier , Kohn Donald B. , Fischer Alain , de Villartay Jean-Pierre , Cavazzana-Calvo Marina
Published: November 2002

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation, which is only partially successful in the absence of an HLA genoidentical donor, thus justifying research to find an alternative therapeutic approach. To this end, RAG-2–deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2−/−Sca-1+ selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)–based MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2−/− sublethally irradiated mice (3Gy). Two months later, T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens, allogeneic cells, and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy, a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether, this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2−/− mice, constituting a significant step toward clinical application.
Antimyeloma efficacy of thalidomide in the SCID-hu model
Authors: Yaccoby Shmuel , Johnson Cherie L. , Mahaffey Susan C. , Wezeman Michele J. , Barlogie Bart , Epstein Joshua
Published: November 2002

To determine the mechanism of thalidomide's antimyeloma efficacy, we studied the drug's activity in our severe combined immunodeficiency-human (SCID-hu) host system for primary human myeloma. In this model, tumor cells interact with the human microenvironment to produce typical myeloma manifestations in the hosts, including stimulation of neoangiogenesis. Because mice are not able to metabolize thalidomide efficiently, SCID-hu mice received implants of fetal human liver fragments under the renal capsule in addition to subcutaneous implants of the fetal human bone. Myeloma cell growth in these mice was similar to their growth in hosts without liver implant, as assessed by change in levels of circulating human immunoglobulins and by histologic examinations. Thalidomide given daily by peritoneal injection significantly inhibited myeloma growth in 7 of 8 experiments, each with myeloma cells from a different patient, in hosts implanted with human liver. In contrast, thalidomide exerted an antimyeloma effect only in 1 of 10 mice without liver implants. Microvessel density in the untreated controls was higher than in thalidomide-responsive hosts but not different from nonresponsive ones. Expression of vascular endothelial growth factor by myeloma cells and by other cells in the human bone, determined immunohistochemically, was not affected by thalidomide treatment in any experiment. Our study suggests that thalidomide metabolism is required for its antimyeloma efficacy. Although response to thalidomide was strongly associated with decreased microvessel density, we were unable to conclude whether reduced microvessel density is a primary result of thalidomide's antiangiogenic activity or is secondary to a lessened tumor burden.
Skewed T-cell receptor repertoire, decreased thymic output, and predominance of terminally differentiated T cells in ataxia telangiectasia
Authors: Giovannetti Antonello , Mazzetta Francesca , Caprini Elisabetta , Aiuti Alessandro , Marziali Marco , Pierdominici Marina , Cossarizza Andrea , Chessa Luciana , Scala Enrico , Quinti Isabella , Russo Giandomenico , Fiorilli Massimo
Published: November 2002

Ataxia telangiectasia (A-T), a genetic disorder caused by the homozygous mutation of the ATM gene, frequently associates with variable degrees of cellular and humoral immunodeficiency. However, the immune defects occurring in patients with A-T are still poorly characterized. Here we show that the T-cell receptor (TCR) variable β (BV)–chain repertoire of 9 A-T patients was restricted by diffuse expansions of some variable genes prevalently occurring within the CD4 subset and clustering to certain TCRBV genes (eg, 5.1, 11, 14, and 23). In addition, the study of the third complementarity-determining region (CDR3) showed, in all patients, significantly altered profiles in most BV genes examined suggesting diffuse oligoclonal expansions. The sequencing of TCR CDR3 regions revealed completely normal V(D)J coding joints and confirmed a reduced diversity of the antigen-receptor repertoire. The B-cell repertoire was similarly restricted and skewed by diffuse oligoclonal expansions with normal V(D)J joints. Thymic output, evaluated by measuring TCR rearrangement excision circles, was extremely low. The majority of peripheral T cells had the phenotype and the function of effector memory cells, indicating that in vivo they are able to respond normally by terminal differentiation to antigenic stimulation. These results indicate that ATM mutation limits the generation of a wide repertoire of normally functioning T and B cells.
Tumor suppression induced by intratumor administration of adenovirus vector expressing NK4, a 4-kringle antagonist of hepatocyte growth factor, and naive dendritic cells
Authors: Kikuchi Toshiaki , Maemondo Makoto , Narumi Koh , Matsumoto Kunio , Nakamura Toshikazu , Nukiwa Toshihiro
Published: November 2002

NK4, a 4-kringle antagonist of hepatocyte growth factor (HGF), is a potent inhibitor of tumor angiogenesis and functions independently of its HGF-antagonistic activity. We have shown previously that in vivo genetic modification of tumors with an adenovirus vector that expresses NK4 (AdNK4) restrains tumor angiogenesis and slows the rate of tumor growth in vivo. In the present study, we investigated the hypothesis that this can be made more efficient by also administering bone marrow–generated dendritic cells (DCs) to the tumor. The data show that the growth of mouse subcutaneous tumors is significantly suppressed by direct administration of DCs into established tumors that had been pretreated with AdNK4 3 days previously. The synergistic antitumor effect produced by the combination therapy of AdNK4 with DCs correlated with the in vivo priming of tumor-specific cytotoxic T lymphocytes. Analysis of mice treated with fluorescence-labeled DCs suggested that DCs injected into the flank tumor could migrate to lymphoid organs in vivo for activation of immune-relevant processes. Knockout mice experiments demonstrated that the tumor regression produced by this combination therapy depends on both major histocompatibility complex (MHC) class I antigen presentation of DCs injected into the tumors and CD8+ T cells of the treated host. Finally, a mechanism for this synergism was suggested by the histological observation that tumor necrosis and apoptosis were induced by genetic engineering of the tumors to express NK4. These findings should be useful in designing novel strategies that use a combination of 2 monotherapies directed against the vascular and immune systems for cancer therapy.
Enhanced splenomegaly and severe liver inflammation in haptoglobin/hemopexin double-null mice after acute hemolysis
Authors: Tolosano Emanuela , Fagoonee Sharmila , Hirsch Emilio , Berger Franklin G. , Baumann Heinz , Silengo Lorenzo , Altruda Fiorella
Published: November 2002

Intravascular hemolysis is associated with several pathologic conditions that include hemoglobinopathies, trauma, malaria, and bacterial infections. Among plasma-protective proteins against oxidative damage caused by red blood cell rupture, haptoglobin and hemopexin are thought to play a crucial role. Haptoglobin and hemopexin, by binding with high-affinity hemoglobin and heme, respectively, exert an antioxidant action by preventing heme-catalyzed free radical production. Moreover, these proteins prevent iron loss by inhibiting glomerular filtration of hemoglobin and heme diffusion through plasma membranes. Analysis of single-null mice demonstrated the antioxidant action of haptoglobin and hemopexin in vivo and suggests that the 2 proteins cooperate in the resolution of hemolytic stress. To evaluate the physiological relevance of the haptoglobin-hemopexin system and the principal targets of its action, we generated haptoglobin-hemopexin double-knockout mice and analyzed them under basal conditions and after acute hemolysis. Whereas haptoglobin-hemopexin double-null mice displayed no obvious alteration in phenotype under basal conditions, nonlethal hemolytic stress in these animals led to pronounced splenomegaly as well as liver inflammation and fibrosis. These data demonstrate that haptoglobin and hemopexin together are essential for protection from splenomegaly and liver fibrosis resulting from intravascular hemolysis.
Stressed apoptotic tumor cells stimulate dendritic cells and induce specific cytotoxic T cells
Authors: Feng Hanping , Zeng Yi , Graner Michael W. , Katsanis Emmanuel
Published: November 2002

We have previously reported that stressed apoptotic tumor cells are more immunogenic in vivo than nonstressed ones. Using confocal microscopy we have confirmed our previous observation that heat-stressed apoptotic 12B1-D1 leukemia cells(BCR-ABL+) express HSP60 and HSP72 on their surface. To explore how the immune system distinguishes stressed from nonstressed apoptotic tumor cells, we analyzed the responses of dendritic cells to these 2 types of apoptotic cells. We found that nonstressed and heat-stressed apoptotic 12B1-D1 cells were taken up by dendritic cells in a comparable fashion. However, when stressed apoptotic 12B1-D1 cells were coincubated with immature dendritic cells for 24 hours, this resulted in greater up-regulation of costimulatory molecules (CD40, CD80, and CD86) on the surface of dendritic cells. Moreover, stressed apoptotic 12B1-D1 cells were more effective in stimulating dendritic cells to secrete interleukin-12 (IL-12) and in enhancing their immunostimulatory functions in mixed leukocyte reactions. Furthermore, we demonstrated that immunization of mice with stressed apoptotic 12B1-D1 cells induced the secretion of T helper-1 (TH1) profile of cytokines by spleen cells. Splenocytes from mice immunized with stressed apoptotic cells, but not nonstressed ones, were capable of lysing 12B1-D1 and the parental 12B1 line, but not a B-cell leukemia line, A20. Our data indicate that stressed apoptotic tumor cells are capable of providing the necessary danger signals, likely through increased surface expression of heat shock proteins (HSPs), resulting in activation/maturation of dendritic cells and, ultimately, the generation of potent antitumor T-cell responses.
Estrogen stimulates arachidonoylethanolamide release from human endothelial cells and platelet activation
Authors: Maccarrone Mauro , Bari Monica , Battista Natalia , Finazzi-Agrò Alessandro
Published: November 2002

Estrogen replacement therapy has been associated with reduction of cardiovascular events in postmenopausal women, though the mechanism for this benefit remains unclear. Here we show that at physiological concentrations estrogen activates the anandamide membrane transporter of human endothelial cells and leads to rapid elevation of calcium (apparent within 5 minutes) and release of nitric oxide (within 15 minutes). These effects are mediated by estrogen binding to a surface receptor, which shows an apparent dissociation constant (Kd) of 9.4 ± 1.4 nM, a maximum binding (Bmax) of 356 ± 12 fmol × mg protein−1, and an apparent molecular mass of approximately 60 kDa. We also show that estrogen binding to surface receptors leads to stimulation of the anandamide-synthesizing enzyme phospholipase D and to inhibition of the anandamide-hydrolyzing enzyme fatty acid amide hydrolase, the latter effect mediated by 15-lipoxygenase activity. Because the endothelial transporter is shown to move anandamide across the cell membranes bidirectionally, taken together these data suggest that the physiological activity of estrogen is to stimulate the release, rather than the uptake, of anandamide from endothelial cells. Moreover, we show that anandamide released from estrogen-stimulated endothelial cells, unlike estrogen itself, inhibits the secretion of serotonin from adenosine diphosphate (ADP)–stimulated platelets. Therefore, it is suggested that the peripheral actions of anandamide could be part of the molecular events responsible for the beneficial effects of estrogen.
Increased hepatic iron in mice lacking classical MHC class I molecules
Authors: Cardoso Elsa M. , Macedo Maria G. , Rohrlich Pierre , Ribeiro Eduarda , Silva Manuel T. , Lemonnier François A. , de Sousa Maria
Published: November 2002

Iron accumulation in the liver in hereditary hemochromatosis (HH) has been shown to be highly variable. Some studies point to the importance of major histocompatibility complex (MHC) class I (MHC-I) and CD8+ cells as modifiers of iron overload. In this report, using mice knockout for H2Kb−/−and H2Db−/− genes, it is demonstrated that lack of classical MHC-I molecules results in a spontaneous increase of nonheme iron content in the liver (mainly located in the hepatocytes) when compared to wild-type mice. In CD8−/−and Rag2−/− mice, no spontaneous hepatic iron accumulation was observed. These results demonstrate for the first time that classical MHC-I molecules could be involved in the regulation of iron metabolism and contribute to the established genotype/phenotype discrepancies seen in HH.
Inefficient response of T lymphocytes to glycosylphosphatidylinositol anchor–negative cells: implications for paroxysmal nocturnal hemoglobinuria
Authors: Murakami Yoshiko , Kosaka Hiroshi , Maeda Yusuke , Nishimura Jun-ichi , Inoue Norimitsu , Ohishi Kazuhito , Okabe Masaru , Takeda Junji , Kinoshita Taroh
Published: November 2002

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder in which clonal cells defective in glycosylphosphatidylinositol (GPI) biosynthesis are expanded, leading to complement-mediated hemolysis. PNH is often associated with bone marrow suppressive conditions, such as aplastic anemia. One hypothetical mechanism for the clonal expansion of GPI−cells in PNH is that the mutant cells escape attack by autoreactive cytotoxic cells that are thought to be responsible for aplastic anemia. Here we studied 2 model systems. First, we made pairs of GPI+ and GPI− EL4 cells that expressed major histocompatibility complex (MHC) class II molecules and various types of ovalbumin. When the GPI-anchored form of ovalbumin was expressed on GPI+ and GPI− cells, only the GPI+cells presented ovalbumin to ovalbumin-specific CD4+ T cells, indicating that if a putative autoantigen recognized by cytotoxic cells is a GPI-anchored protein, GPI− cells are less sensitive to cytotoxic cells. Second, antigen-specific as well as alloreactive CD4+ T cells responded less efficiently to GPI− than GPI+ cells in proliferation assays. In vivo, when GPI− and GPI+ fetal liver cells, and CD4+ T cells alloreactive to them, were cotransplanted into irradiated hosts, the contribution of GPI− cells in peripheral blood cells was significantly higher than that of GPI+ cells. The results obtained with the second model suggest that certain GPI-anchored protein on target cells is important for recognition by T cells. These results provide the first experimental evidence for the hypothesis that GPI− cells escape from immunologic attack.