Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation

Liver-specific and nonliver-specific methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (AdoMet), the principal biological methyl donor. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and is induced during liver growth and dedifferentiation. To examine the influence of MAT1A on hepatic growth, we studied the effects of a targeted disruption of the murine MAT1A gene. MAT1A mRNA and protein levels were absent in homozygous knockout mice. At 3 months, plasma methionine level increased 776% in knockouts. Hepatic AdoMet and glutathione levels were reduced by 74 and 40%, respectively, whereas S-adenosylhomocysteine, methylthioadenosine, and global DNA methylation were unchanged. The body weight of 3-month-old knockout mice was unchanged from wild-type littermates, but the liver weight was increased 40%. The Affymetrix genechip system and Northern and Western blot analyses were used to analyze differential expression of genes. The expression of many acute phase-response and inflammatory markers, including orosomucoid, amyloid, metallothionein, Fas antigen, and growth-related genes, including early growth response 1 and proliferating cell nuclear antigen, is increased in the knockout animal. At 3 months, knockout mice are more susceptible to choline-deficient diet-induced fatty liver. At 8 months, knockout mice developed spontaneous macrovesicular steatosis and predominantly periportal mononuclear cell infiltration. Thus, absence of MAT1A resulted in a liver that is more susceptible to injury, expresses markers of an acute phase response, and displays increased proliferation.

T cell-specific FADD-deficient mice: FADD is required for early T cell development

FADD/Mort1, initially identified as a Fas-associated death-domain containing protein, functions as an adapter molecule in apoptosis initiated by Fas, tumor necrosis factor receptor-I, DR3, and TRAIL-receptors. However, FADD likely participates in additional signaling cascades. FADD-null mutations in mice are embryonic-lethal, and analysis of FADD−/− T cells from RAG-1−/− reconstituted chimeras has suggested a role for FADD in proliferation of mature T cells. Here, we report the generation of T cell-specific FADD-deficient mice via a conditional genomic rescue approach. We find that FADD-deficiency leads to inhibition of T cell development at the CD4−CD8− stage and a reduction in the number of mature T cells. The FADD mutation does not affect apoptosis or the proximal signaling events of the pre-T cell receptor; introduction of a T cell receptor transgene fails to rescue the mutant phenotype. These data suggest that FADD, through either a death-domain containing receptor or a novel receptor-independent mechanism, is required for the proliferative phase of early T cell development.

A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation

We have identified a human cytomegalovirus cell-death suppressor, denoted vICA, encoded by the viral UL36 gene. vICA inhibits Fas-mediated apoptosis by binding to the pro-domain of caspase-8 and preventing its activation. vICA does not share significant sequence homology with FLIPs or other known suppressors of apoptosis, suggesting that this protein represents a new class of cell-death suppressors. Notably, resistance to Fas-mediated apoptosis is delayed in fibroblasts infected with viruses that encode mutant vICA, suggesting that vICA suppresses death-receptor-induced cell death in the context of viral infection. Although vICA is dispensable for viral replication in vitro, the common targeting of caspase-8 activation by diverse herpesviruses argues for an important role for this antiapoptotic mechanism in the pathogenesis of viral infection in the host, most likely in avoiding immune clearance by cytotoxic lymphocytes and natural killer cells.

RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways.

With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor. Both of these proteins induce apoptosis in mammalian cells. Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis. However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene. By contrast, FADD's ability to induce cell death does not depend on crosslinking. Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD. Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD. Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases.

In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains.

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways.

Expression of galectin-3 modulates T-cell growth and apoptosis.

Galectin-3 is a member (if a large family of beta-galactoside-binding animal lectins. It has been shown that the expression of galectin-3 is upregulated in proliferating cells, suggesting a possible role for this lectin in regulation of cell growth. Previously, we have shown that T cells infected with human T-cell leukemia virus type I express high levels of galectin-3, in contrast to uninfected cells, which do not express detectable amounts of this protein. In this study, we examined growth properties of human leukemia T cells transfected with galectin-3 cDNA, and thus constitutively overexpressing this lectin. Transfectants expressing galectin-3 displayed higher growth rates than control transfectants, which do not express this lectin. Furthermore, galectin-3 expression in these cells confers resistance to apoptosis induced by anti-Fas antibody and staurosporine. Galectin-3 was found to have significant sequence similarity with Bcl-2, a well-characterized suppressor of apoptosis. In particular, the lectin contains the NWGR motif that is highly conserved among members of the Bcl-2 family and shown to be critical for the apoptosis-suppressing activity. We further demonstrated that galectin-3 interacts with Bc1-2 in a lactose-inhibitable manner. We conclude that galectin-3 is a regulator of cell growth and apoptosis and it may function through a cell death inhibition pathway that involves Bcl-2.

Differential effects of staphylococcal toxic shock syndrome toxin-1 on B cell apoptosis.

Superantigens, such as toxic shock syndrome toxin 1 (TSST-1), have been implicated in the pathogenesis of several autoimmune and allergic diseases associated with polyclonal B cell activation. In this report, we studied the in vitro effects of TSST-1 on B cell activation. We show herein that TSST-1 produced antagonistic effects on Ig synthesis by peripheral blood mononuclear cells (PBMC) from normal subjects, depending on the concentration used; Ig production was inhibited at 1000 pg/ml (P < 0.01) and enhanced at 1 and 0.01 pg/ml (P < 0.01) of toxin. Cultures of PBMC were then examined for morphologic features and DNA fragmentation characteristic for apoptosis. B cells exhibited a significantly higher (P < 0.01) incidence of apoptosis after stimulation with 1000 pg/ml of TSST-1 compared with 1 or 0.01 pg/ml of toxin or medium alone. Abundant expression of Fas, a cell surface protein that mediates apoptosis, was detected on B cells after stimulation with 1000 pg/ml of TSST-1 and was significantly higher on B cells undergoing apoptosis than on live cells (P = 0.01). Additionally, increased Fas expression and B cell death occurred at concentrations of TSST-1 inducing the production of high amounts of gamma interferon (IFN-gamma), and both events could be blocked by neutralizing anti-IFN-gamma antibody. These findings suggest that high concentrations of TSST-1 can induce IFN-gamma-dependent B cell apoptosis, whereas at low concentrations it stimulates Ig synthesis by PBMC from normal subjects. These findings support the concept that staphylococcal toxins have a role in B cell hyperactivity in autoimmunity and allergy.

Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.

Fasciclin II controls proneural gene expression in Drosophila.

Fasciclin II (Fas II), an NCAM-like cell adhesion molecule in Drosophila, is expressed on a subset of embryonic axons and controls selective axon fasiculation. Fas II is also expressed in imaginal discs. Here we use genetic analysis to show that Fas II is required for the control of proneural gene expression. Clusters of cells in the eye-antennal imaginal disc express the achaete proneural gene and give rise to mechanosensory neurons; other clusters of cells express the atonal gene and give rise to ocellar photoreceptor neurons. In fasII loss-of-function mutants, the expression of both proneural genes is absent in certain locations, and, as a result, the corresponding sensory precursors fail to develop. In fasII gain-of-function conditions, extra sensory structures arise from this same region of the imaginal disc. Mutations in the Abelson tyrosine kinase gene show dominant interactions with fasII mutations, suggesting that Abl and Fas II function in a signaling pathway that controls proneural gene expression.

Human fatty acid synthase: properties and molecular cloning.

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.

Hybrid convex combinations for IIR system identification.

The low complexity of IIR adaptive filters (AFs) is specially appealing to realtime applications but some drawbacks have been preventing their widespread use so far. For gradient based IIR AFs, adverse operational conditions cause convergence problems in system identification scenarios: underdamped and clustered poles, undermodelling or non-white input signals lead to error surfaces where the adaptation nearly stops on large plateaus or get stuck at sub-optimal local minima that can not be identified as such a priori. Furthermore, the non-stationarity in the input regressor brought by the filter recursivity and the approximations made by the update rules of the stochastic gradient algorithms constrain the learning step size to small values, causing slow convergence. In this work, we propose IIR performance enhancement strategies based on hybrid combinations of AFs that achieve higher convergence rates than ordinary IIR AFs while keeping the stability.

How do primary health care teams learn to integrate intimate partner violence (IPV) management? A realist evaluation protocol

Background: Despite the existence of ample literature dealing, on the one hand, with the integration of innovations within health systems and team learning, and, on the other hand, with different aspects of the detection and management of intimate partner violence (IPV) within healthcare facilities, research that explores how health innovations that go beyond biomedical issues—such as IPV management—get integrated into health systems, and that focuses on healthcare teams’ learning processes is, to the best of our knowledge, very scarce if not absent. This realist evaluation protocol aims to ascertain: why, how, and under what circumstances primary healthcare teams engage (if at all) in a learning process to integrate IPV management in their practices; and why, how, and under what circumstances team learning processes lead to the development of organizational culture and values regarding IPV management, and the delivery of IPV management services. Methods: This study will be conducted in Spain using a multiple-case study design. Data will be collected from selected cases (primary healthcare teams) through different methods: individual and group interviews, routinely collected statistical data, documentary review, and observation. Cases will be purposively selected in order to enable testing the initial middle-range theory (MRT). After in-depth exploration of a limited number of cases, additional cases will be chosen for their ability to contribute to refining the emerging MRT to explain how primary healthcare learn to integrate intimate partner violence management. Discussion: Evaluations of health sector responses to IPV are scarce, and even fewer focus on why, how, and when the healthcare services integrate IPV management. There is a consensus that healthcare professionals and healthcare teams play a key role in this integration, and that training is important in order to realize changes. However, little is known about team learning of IPV management, both in terms of how to trigger such learning and how team learning is connected with changes in organizational culture and values, and in service delivery. This realist evaluation protocol aims to contribute to this knowledge by conducting this project in a country, Spain, where great endeavours have been made towards the integration of IPV management within the health system.

Mapping and exploring health systems' response to intimate partner violence in Spain

Background: For a comprehensive health sector response to intimate partner violence (IPV), interventions should target individual and health facility levels, along with the broader health systems level which includes issues of governance, financing, planning, service delivery, monitoring and evaluation, and demand generation. This study aims to map and explore the integration of IPV response in the Spanish national health system. Methods: Information was collected on five key areas based on WHO recommendations: policy environment, protocols, training, monitoring and prevention. A systematic review of public documents was conducted to assess 39 indicators in each of Spain’s 17 regional health systems. In addition, we performed qualitative content analysis of 26 individual interviews with key informants responsible for coordinating the health sector response to IPV in Spain. Results: In 88% of the 17 autonomous regions, the laws concerning IPV included the health sector response, but the integration of IPV in regional health plans was just 41%. Despite the existence of a supportive national structure, responding to IPV still relies strongly on the will of health professionals. All seventeen regions had published comprehensive protocols to guide the health sector response to IPV, but participants recognized that responding to IPV was more complex than merely following the steps of a protocol. Published training plans existed in 43% of the regional health systems, but none had institutionalized IPV training in medical and nursing schools. Only 12% of regional health systems collected information on the quality of the IPV response, and there are many limitations to collecting information on IPV within health services, for example underreporting, fears about confidentiality, and underuse of data for monitoring purposes. Finally, preventive activities that were considered essential were not institutionalized anywhere. Conclusions: Within the Spanish health system, differences exist in terms of achievements both between regions and between the areas assessed. Progress towards integration of IPV has been notable at the level of policy, less outstanding regarding health service delivery, and very limited in terms of preventive actions.

La respuesta sanitaria a la violencia de género. ¿Adaptar el sistema o medicalizar la violencia? estudio cualitativo

Antecedentes/Objetivos: El sistema sanitario puede jugar un papel clave en la prevención y atención de la violencia de género (VG), debido a las consecuencias para la salud de la violencia y al hecho de que las mujeres afectadas acceden a los servicios de salud con mayor frecuencia que a otros servicios públicos. El objetivo de este estudio es analizar cómo entienden los profesionales encargados de la coordinación de los programas de VG en los servicios regionales de salud la integración de la respuesta sanitaria a este problema en el Sistema Nacional de Salud español. Métodos: Estudio cualitativo en el que se realizaron 26 entrevistas individuales a informantes clave del nivel gerencial, 23 de las 17 comunidades autónomas y 3 del nivel nacional, entre julio de 2012 a marzo de 2013. Las transcripciones se importaron al software informático Atlas.ti-5 y se analizaron de acuerdo al método de la Teoría Fundamentada. Resultados: Se desarrolló un modelo conceptual que explica los esfuerzos y retos cuando se intenta integrar la respuesta a la VG en el sistema sanitario, que permanece fuertemente biomedicalizado. Emergieron una categoría central: Integrando respuestas a problemas no biomédicos en sistemas de salud biomédicos, el caso de la VG; y 4 categorías relacionadas con la central: La VG es un problema complejo que genera resistencia o activismo; El mandato para integrar una respuesta sanitaria a la violencia: una prioridad no siempre priorizada; El sistema sanitario español, respetuoso con la autonomía de los profesionales, biomédico y familiarista; y Desarrollando respuestas a la integración de la violencia: adaptar el sistema de salud o medicalizar la violencia. Conclusiones: Integrar el abordaje de la VG en un sistema de salud biomédico es un reto. Los hallazgos sugieren que la motivación individual puede compensar las deficiencias en el sistema existente, en términos de ser más centrada en la persona y, por lo tanto, fomentar una fuerte relación de confianza. Sin embargo, la sostenibilidad de los procesos dependientes en la motivación e interés individual es difícil si no hay una estructura organizativa que los respalde.

Estudio cualitativo sobre la sostenibilidad de los programas de violencia de género en el sistema sanitario español durante la crisis

Antecedentes/Objetivos: La Ley 1/2004 de atención a la integral a la violencia de género institucionaliza la respuesta sanitaria a este problema. En la actualidad, los servicios sanitarios se están viendo afectados por las políticas de austeridad del gobierno como respuesta a la crisis financiera, lo que puede afectar negativamente a la atención de la violencia de género. El objetivo de este estudio es explorar las percepciones de profesionales sanitarios del nivel gerencial sobre el impacto de la crisis económica en la respuesta sanitaria a la violencia de género en España. Métodos: Estudio cualitativo con 26 entrevistas individuales a informantes clave del nivel gerencial, 23 de las 17 Comunidades Autónomas y 3 del nivel nacional, entre julio de 2012 a marzo de 2013. Las transcripciones se importaron al software informático Atlas.ti-5 y se realizó un análisis de contenido cualitativo. Resultados: Se identificaron 4 categorías que explican las consecuencias de las políticas de austeridad en la atención sanitaria de la violencia de género. Tres de ellas hacen referencia a los efectos negativos: Desmotivación y pesimismo para avanzar en la integración de la respuesta a la violencia; Un sistema desbordado por los recortes y presión asistencial; y Necesidad de una mayor implicación por parte del personal sanitario para suplir las carencias del sistema. Una cuarta categoría concibe la crisis como oportunidad: El desafío es mantener y no desmantelar, una visión optimista de la crisis como un reto para afianzar lo que hay. Las personas implicadas luchan para mantener el abordaje de la violencia en las agendas, movidas por su motivación personal, pero sienten que es luchar contra un muro o ir contracorriente porque no se ha producido una integración oficial en la práctica sino sólo en la teoría, a raíz de la ley 1/2004 y las políticas de igualdad impulsadas por el anterior gobierno. Todo ello se ve dificultado por la desaparición de las subvenciones estatales para la formación y sensibilización, implantación de protocolos o creación de sistemas de información, junto a que no está en la cartera de servicios de las comunidades. Conclusiones: Continuar con la integración del abordaje de la violencia de género en el sistema de salud en época de crisis no es una prioridad. Los hallazgos sugieren que la motivación individual trata de compensar las deficiencias en el sistema existente, pero que el voluntarismo de las personas individuales no es suficiente sin estructuras organizativas que les respalden, lo que se ve especialmente dificultado en tiempos de crisis.