Defective TNF-alpha-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.

Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz.

We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli. We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (G') and loss modulus (G") from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field. Under baseline conditions, G' increased weakly with frequency, whereas G" was independent of the frequency. The cell was predominantly elastic, with the ratio of G" to G' (defined as eta) being ~0.35 at all frequencies. G' and G" increased together after contractile activation and decreased together after deactivation, whereas eta remained unaltered in each case. Thus elastic and dissipative stresses were coupled during changes in contractile activation. G' and G" decreased with disruption of the actin fibers by cytochalasin D, but eta increased. These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton.

Capital humano local y productividad en las provincias españolas

En la última década, distintos estudios han intentado contrastar empíricamente la existencia de una relación entre el stock de capital humanolocal y la productividad del territorio, así como la posible presencia de economías externas asociadas a aquél. El resultado común de dichos estudios ha consistido en encontrar una correlación positiva entre ambas variables Losdiversos autores no coinciden, en cambio, a la hora de explicar dicho resultado: un primer grupo de autores argumenta la presencia de economíasexternas vinculadas al capital humano mientras que un segundo grupo plantea la existencia de relaciones de complementariedad entre los diversos factores productivos y, más en concreto, entre el capital humano y el capital físico.El objetivo de este trabajo es analizar la existencia de una posible relación positiva entre el nivel de capital humano de las provincias españolas y su productividad de éstas y, a continuación, averiguar si el canal a través delcual se produce el efecto son las economías externas. Para ello, se aplica unametodología que consta de dos etapas. En la primera, se estima una ecuación de Mincer utilizando información de la Encuesta de Presupuestos Familiares a fin de obtener una estimación de la productividad media de cada una de las provincias españolas una vez controlado el efecto del capital humano de los individuos sobre su propia productividad. En una segunda etapa, la estimación de la productividad provincial media estimada se introduce como variable endógena en una nueva ecuación cuyas variables explicativas intentan aproximar el nivel de capital humano de cada una de las provincias. A partir de esta segunda regresión se detecta una relación positiva entre la productividad media estimada del territorio y el nivel educativo medio delmismo. Sin embargo, la principal conclusión del análisis realizado es que dicha relación no puede explicarse por el impacto de las economías externas generadas exógenamente por el capital humano, sino que debe atribuirse a otros efectos que, actuando también por lado de la demanda, impulsen al alza la productividad.

Testing for hysteresis in unemployment in OECD countries: new evidence using stationarity panel tests with breaks

This paper tests hysteresis effects in unemployment using panel data for 19 OECD countries covering the period 1956-2001. The tests exploit the cross-section variations of the series, and additionally, allow for a diferent number of endogenous breakpoints in the unemployment series. The critical values are simulated based on our specific panel sizes and time periods. The findings stress the importance of accounting for exogenous shocks in the series and give support to the natural-rate hypothesis of unemployment for the majority of the countries analyzed

Testing for hysteresis in unemployment in OECD countries: new evidence using stationarity panel tests with breaks

This paper tests hysteresis effects in unemployment using panel data for 19 OECD countries covering the period 1956-2001. The tests exploit the cross-section variations of the series, and additionally, allow for a diferent number of endogenous breakpoints in the unemployment series. The critical values are simulated based on our specific panel sizes and time periods. The findings stress the importance of accounting for exogenous shocks in the series and give support to the natural-rate hypothesis of unemployment for the majority of the countries analyzed

Capital humano local y productividad en las provincias españolas

En la última década, distintos estudios han intentado contrastar empíricamente la existencia de una relación entre el stock de capital humanolocal y la productividad del territorio, así como la posible presencia de economías externas asociadas a aquél. El resultado común de dichos estudios ha consistido en encontrar una correlación positiva entre ambas variables Losdiversos autores no coinciden, en cambio, a la hora de explicar dicho resultado: un primer grupo de autores argumenta la presencia de economíasexternas vinculadas al capital humano mientras que un segundo grupo plantea la existencia de relaciones de complementariedad entre los diversos factores productivos y, más en concreto, entre el capital humano y el capital físico.El objetivo de este trabajo es analizar la existencia de una posible relación positiva entre el nivel de capital humano de las provincias españolas y su productividad de éstas y, a continuación, averiguar si el canal a través delcual se produce el efecto son las economías externas. Para ello, se aplica unametodología que consta de dos etapas. En la primera, se estima una ecuación de Mincer utilizando información de la Encuesta de Presupuestos Familiares a fin de obtener una estimación de la productividad media de cada una de las provincias españolas una vez controlado el efecto del capital humano de los individuos sobre su propia productividad. En una segunda etapa, la estimación de la productividad provincial media estimada se introduce como variable endógena en una nueva ecuación cuyas variables explicativas intentan aproximar el nivel de capital humano de cada una de las provincias. A partir de esta segunda regresión se detecta una relación positiva entre la productividad media estimada del territorio y el nivel educativo medio delmismo. Sin embargo, la principal conclusión del análisis realizado es que dicha relación no puede explicarse por el impacto de las economías externas generadas exógenamente por el capital humano, sino que debe atribuirse a otros efectos que, actuando también por lado de la demanda, impulsen al alza la productividad.

Interiority of the optimal population growth rate with endogenous fertility

This paper analyzes the issue of the interiority of the optimal population growth rate in a two-period overlapping generations model with endogenous fertility. Using Cobb-Douglas utility and production functions, we show that the introduction of a cost of raising children allows for the possibility of the existence of an interior global maximum in the planner¿s problem, contrary to the exogenous fertility case

Spectral analysis of the luteinizing hormone in the blood samples

Generally, medicine books are concentrated almost exclusively in explaining methodology that analyzes fixed measures, measures done in a certain moment, nevertheless the evolution of the measurement and correct interpretation of the missed values are very important and sometimes can give the key information of the results obtained. Thus, the analysis of the temporary series and spectral analysis or analysis of the time series in the dominion of frequencies can be regarded as an appropriate tool for this kind of studies.In this work the frequency of the pulsating secretion of luteinizing hormone LH (thatregulates the fertile life of women) were analyzed in order to determine the existence of the significant frequencies obtained by analysis of Fourier. Detection of the frequencies, with which the pulsating secretion of the LH takes place, is a quite difficult question due topresence of the random errors in measures and samplings, i.e. that pulsating secretions of small amplitude are not detected and disregarded. In physiology it is accepted that cyclical patterns in the secretion of the LH exist and in the results of this research confirm this pattern and determine its frequency presented in the corresponded periodograms to each of studied cycle. The obtained results can be used as key pattern for future sampling frequencies in order to ¿catch¿ the significant picks of the luteinizing hormone and reflect on time forproductivity treatment of women.

Anti-inflammatory effects of pancreatitis associated protein in inflammatory bowel disease

Background and aims: Increased pancreatitis associated protein (PAP) mRNA has been reported in active inflammatory bowel disease (IBD). The aims of the current study were to characterise PAP production in IBD and the effects of PAP on inflammation. Patients and methods: Serum PAP levels were determined in healthy controls (n¿=¿29), inflammatory controls (n¿=¿14), and IBD patients (n¿=¿171). Ex vivo PAP secretion in intestinal tissue was measured in 56 IBD patients and 13 healthy controls. Cellular origin of PAP was determined by immunohistochemistry. The effects of exogenous PAP on nuclear factor ¿B (NF¿B) activation, proinflammatory cytokine production, and endothelial adhesion molecule expression were also analysed ex vivo. Results: Patients with active IBD had increased serum PAP levels compared with controls, and these levels correlated with clinical and endoscopic disease severity. Ex vivo intestinal PAP synthesis was increased in active IBD and correlated with endoscopic and histological severity of inflammatory lesions. PAP localised to colonic Paneth cells. Incubation of mucosa from active Crohn¿s disease with PAP dose dependently reduced proinflammatory cytokines secretion. PAP prevented TNF-¿ induced NF¿B activation in monocytic, epithelial, and endothelial cells and reduced proinflammatory cytokine mRNA levels and adhesion molecule expression. Conclusions: PAP is synthesised by Paneth cells and is overexpressed in colonic tissue of active IBD. PAP inhibits NF¿B activation and downregulates cytokine production and adhesion molecule expression in inflamed tissue. It may represent an anti-inflammatory mechanism and new therapeutic strategy in IBD.

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4¿dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.

Interiority of the optimal population growth rate with endogenous fertility

This paper analyzes the issue of the interiority of the optimal population growth rate in a two-period overlapping generations model with endogenous fertility. Using Cobb-Douglas utility and production functions, we show that the introduction of a cost of raising children allows for the possibility of the existence of an interior global maximum in the planner¿s problem, contrary to the exogenous fertility case

Fish Glucose Transporter (GLUT)-4 Differs from Rat GLUT4 in Its Traffic Characteristics but Can Translocate to the Cell Surface in Response to Insulin in Skeletal Muscle Cells

In mammals, glucose transporter (GLUT)-4 plays an important role in glucose homeostasis mediating insulin action to increase glucose uptake in insulin-responsive tissues. In the basal state, GLUT4 is located in intracellular compartments and upon insulin stimulation is recruited to the plasma membrane, allowing glucose entry into the cell. Compared with mammals, fish are less efficient restoring plasma glucose after dietary or exogenous glucose administration. Recently our group cloned a GLUT4-homolog in skeletal muscle from brown trout (btGLUT4) that differs in protein motifs believed to be important for endocytosis and sorting of mammalian GLUT4. To study the traffic of btGLUT4, we generated a stable L6 muscle cell line overexpressing myc-tagged btGLUT4 (btGLUT4myc). Insulin stimulated btGLUT4myc recruitment to the cell surface, although to a lesser extent than rat-GLUT4myc, and enhanced glucose uptake. Interestingly, btGLUT4myc showed a higher steady-state level at the cell surface under basal conditions than rat-GLUT4myc due to a higher rate of recycling of btGLUT4myc and not to a slower endocytic rate, compared with rat-GLUT4myc. Furthermore, unlike rat-GLUT4myc, btGLUT4myc had a diffuse distribution throughout the cytoplasm of L6 myoblasts. In primary brown trout skeletal muscle cells, insulin also promoted the translocation of endogenous btGLUT4 to the plasma membrane and enhanced glucose transport. Moreover, btGLUT4 exhibited a diffuse intracellular localization in unstimulated trout myocytes. Our data suggest that btGLUT4 is subjected to a different intracellular traffic from rat-GLUT4 and may explain the relative glucose intolerance observed in fish.

Histology of Organogenic and Embryogenic Responses in Cotyledons of Somatic Embryos of Quercus Suber L.

In cork oak (Quercus suber L.), recurrent embryogenesis is produced in vitro through autoembryony without exogenous plant growth regulators (PGRs); secondary embryos appear on the embryo axis but seldom on cotyledons. Focusing mainly on the histological origin of neoformations, we investigated the influence of the embryo axis and exogenous PGRs on the embryogenic potential of somatic embryo cotyledons. Isolated cotyledons of somatic embryos became necrotic when cultured on PGR-free medium but gave secondary embryos when cultured on media containing benzyladenine and naphthaleneacetic acid. Cotyledons of cork oak somatic embryos are competent to give embryogenic responses. Isolated cotyledons without a petiole showed a lower percentage of embryogenic response than did those with a petiole. In petioles, somatic embryos arose from inner parenchyma tissues following a multicellular budding pattern. Joined to the embryo axis, cotyledons did not show morphogenic responses when cultured on PGR-free medium but revealed budlike and phylloid formations when cultured on medium with PGRs. The different morphogenic behavior displayed by somatic cotyledons indicates an influence of the embryo axis and indicates a relationship between organogenic and embryogenic regeneration pathways

Coworker networks in the labour market

This paper studies the role coworker-based networks play for individual labour marketoutcomes. I analyse how the provision of labour market relevant information by formercoworkers affects the employment probabilities and, if hired, the wages of male workerswho have previously become unemployed as the result of an establishment closure. Toidentify the causal effect of an individual worker's network on labour market outcomes, Iexploit exogenous variation in the strength of these networks that is due to the occurrenceof mass-layoffs in the establishments of former coworkers. The empirical analysis is basedon administrative data that comprise the universe of workers employed in Germany between1980 and 2001. The results suggest a strong positive effect of a higher employmentrate in a worker's network of former coworkers on his re-employment probability afterdisplacement: a 10 percentage point increase in the prevailing employment rate in thenetwork increases the re-employment probability by 7.5 percentage points. In contrast,there is no evidence of a statistically significant effect on wages.