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ABSTRACTGliomas, particularly glioblastoma multiforme, perturb the blood-brain barrier and cause brain edema that contributes to morbidity and mortality. The mechanisms underlying this vasogenic edema are poorly understood. We examined the effects of cocultured primary cultured human glioblastoma cells and glioma-derived growth factors on the endothelial cell tight junction proteins claudin 1, claudin 5, occludin, and zonula occludens 1 of brain-derived microvascular endothelial cells and a human umbilical vein endothelial cell line. Cocultured glioblastoma cells and glioma-derived factors (e.g. transforming growth factor β2) enhanced the paracellular flux of endothelial cell monolayers in conjunction with downregulation of the tight junction proteins. Neutralizing anti-transforming growth factor β2 antibodies partially restored the barrier properties in this in vitro blood-brain barrier model. The involvement of endothelial cell-derived matrix metalloproteinases (MMPs) was demonstrated by quantitative reverse-transcriptase-polymerase chain reaction analysis and by the determination of MMP activities via zymography and fluorometry in the presence or absence of the MMP inhibitor GM6001. Occludin, claudin 1, and claudin 5 were expressed in microvascular endothelial cells in nonneoplastic brain samples but were significantly reduced in anaplastic astrocytoma and glioblastoma samples. Taken together, these in vitro and in vivo results indicate that glioma-derived factors may induce MMPs and downregulate endothelial tight junction protein and, thus, play a key role in glioma-induced impairment of the blood-brain barrier.

The Na,K-ATPase hydrolyzes ATP to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane. Here, we report two high-resolution NMR structures of the 213-residue nucleotide-binding domain of rat alpha1 Na,K-ATPase, determined in the absence and the presence of ATP. The nucleotide binds in the anti conformation and shows a relative paucity of interactions with the protein, reflecting the low-affinity ATP-binding state. Binding of ATP induces substantial conformational changes in the binding pocket and in residues located in the hinge region connecting the N- and P-domains. Structural comparison with the Ca-ATPase stabilized by the inhibitor thapsigargin, E2(TG), and the model of the H-ATPase in the E1 form suggests that the observed changes may trigger the series of events necessary for the release of the K+ ions and/or disengagement of the A-domain, leading to the eventual transfer of the gamma-phosphate group to the invariant Asp369.

The functional unit of the Na,K-ATPase consists of a catalytic α subunit noncovalently linked with a glycoprotein subunit, β . Using ouabain binding assays and immunoprecipitation of rodent α /β complexes, we show here that all six possible isozymes between three α and two β isoforms can be formed in Xenopus oocytes. Two isoformspecific differences in α /β interactions are observed: (i) α 1/β 1 and α 2/β 2 complexes, in contrast to α 1/β 2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) β 2 subunits must carry N-glycans to combine with α 1 but not with α 2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated β 1 subunit comprising the ectodomain results in assembly with α 1 and α 2, but not with α 3; the β 2 ectodomain combines with α 2 only. A chimera consisting of 81% and 19% of the α 1 N terminus and α 2 C terminus, respectively, behaves like α 2 and coprecipitates with the β 2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the β 2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase α and β subunits.

Gliomas, particularly glioblastoma multiforme, perturb the blood-brain barrier and cause brain edema that contributes to morbidity and mortality. The mechanisms underlying this vasogenic edema are poorly understood. We examined the effects of cocultured primary cultured human glioblastoma cells and glioma-derived growth factors on the endothelial cell tight junction proteins claudin 1, claudin 5, occludin, and zonula occludens 1 of brain-derived microvascular endothelial cells and a human umbilical vein endothelial cell line. Cocultured glioblastoma cells and glioma-derived factors (e.g. transforming growth factor beta2) enhanced the paracellular flux of endothelial cell monolayers in conjunction with downregulation of the tight junction proteins. Neutralizing anti-transforming growth factor beta2 antibodies partially restored the barrier properties in this in vitro blood-brain barrier model. The involvement of endothelial cell-derived matrix metalloproteinases (MMPs) was demonstrated by quantitative reverse-transcriptase-polymerase chain reaction analysis and by the determination of MMP activities via zymography and fluorometry in the presence or absence of the MMP inhibitor GM6001. Occludin, claudin 1, and claudin 5 were expressed in microvascular endothelial cells in nonneoplastic brain samples but were significantly reduced in anaplastic astrocytoma and glioblastoma samples. Taken together, these in vitro and in vivo results indicate that glioma-derived factors may induce MMPs and downregulate endothelial tight junction protein and, thus, play a key role in glioma-induced impairment of the blood-brain barrier.

Gliomas, particularly glioblastoma multiforme, perturb the blood-brain barrier and cause brain edema that contributes to morbidity and mortality. The mechanisms underlying this vasogenic edema are poorly understood. We examined the effects of cocultured primary cultured human glioblastoma cells and glioma-derived growth factors on the endothelial cell tight junction proteins claudin 1, claudin 5, occludin, and zonula occludens 1 of brain-derived microvascular endothelial cells and a human umbilical vein endothelial cell line. Cocultured glioblastoma cells and glioma-derived factors (e.g. transforming growth factor beta2) enhanced the paracellular flux of endothelial cell monolayers in conjunction with downregulation of the tight junction proteins. Neutralizing anti-transforming growth factor beta2 antibodies partially restored the barrier properties in this in vitro blood-brain barrier model. The involvement of endothelial cell-derived matrix metalloproteinases (MMPs) was demonstrated by quantitative reverse-transcriptase-polymerase chain reaction analysis and by the determination of MMP activities via zymography and fluorometry in the presence or absence of the MMP inhibitor GM6001. Occludin, claudin 1, and claudin 5 were expressed in microvascular endothelial cells in nonneoplastic brain samples but were significantly reduced in anaplastic astrocytoma and glioblastoma samples. Taken together, these in vitro and in vivo results indicate that glioma-derived factors may induce MMPs and downregulate endothelial tight junction protein and, thus, play a key role in glioma-induced impairment of the blood-brain barrier.

Amazonia hosts the Earth’s largest tropical forests and has been shown to be an important carbon sink over recent decades 1 – 3 . This carbon sink seems to be in decline, however, as a result of factors such as deforestation and climate change 1 – 3 . Here we investigate Amazonia’s carbon budget and the main drivers responsible for its change into a carbon source. We performed 590 aircraft vertical profiling measurements of lower-tropospheric concentrations of carbon dioxide and carbon monoxide at four sites in Amazonia from 2010 to 2018 4 . We find that total carbon emissions are greater in eastern Amazonia than in the western part, mostly as a result of spatial differences in carbon-monoxide-derived fire emissions. Southeastern Amazonia, in particular, acts as a net carbon source (total carbon flux minus fire emissions) to the atmosphere. Over the past 40 years, eastern Amazonia has been subjected to more deforestation, warming and moisture stress than the western part, especially during the dry season, with the southeast experiencing the strongest trends 5 – 9 . We explore the effect of climate change and deforestation trends on carbon emissions at our study sites, and find that the intensification of the dry season and an increase in deforestation seem to promote ecosystem stress, increase in fire occurrence, and higher carbon emissions in the eastern Amazon. This is in line with recent studies that indicate an increase in tree mortality and a reduction in photosynthesis as a result of climatic changes across Amazonia 1 , 10 . Aircraft observations of atmospheric carbon dioxide and monoxide concentrations in Brazil show higher carbon emissions in eastern Amazonia than in the western part, which are linked to increased ecosystem stress and fire occurrence.

The Amazon forest carbon sink is declining, mainly as a result of land-use and climate change 1 – 4 . Here we investigate how changes in law enforcement of environmental protection policies may have affected the Amazonian carbon balance between 2010 and 2018 compared with 2019 and 2020, based on atmospheric CO 2 vertical profiles 5 , 6 , deforestation 7 and fire data 8 , as well as infraction notices related to illegal deforestation 9 . We estimate that Amazonia carbon emissions increased from a mean of 0.24 ± 0.08 PgC year −1 in 2010–2018 to 0.44 ± 0.10 PgC year −1 in 2019 and 0.52 ± 0.10 PgC year −1 in 2020 (± uncertainty). The observed increases in deforestation were 82% and 77% (94% accuracy) and burned area were 14% and 42% in 2019 and 2020 compared with the 2010–2018 mean, respectively. We find that the numbers of notifications of infractions against flora decreased by 30% and 54% and fines paid by 74% and 89% in 2019 and 2020, respectively. Carbon losses during 2019–2020 were comparable with those of the record warm El Niño (2015–2016) without an extreme drought event. Statistical tests show that the observed differences between the 2010–2018 mean and 2019–2020 are unlikely to have arisen by chance. The changes in the carbon budget of Amazonia during 2019–2020 were mainly because of western Amazonia becoming a carbon source. Our results indicate that a decline in law enforcement led to increases in deforestation, biomass burning and forest degradation, which increased carbon emissions and enhanced drying and warming of the Amazon forests. Comparison of the carbon balance during 2010–2018 with 2019 and 2020 shows that a decline in law enforcement may have led to an increase in Amazon forest carbon emissions.

Atmospheric methane concentrations were nearly constant between 1999 and 2006, but have been rising since by an average of ~8 ppb per year. Increases in wetland emissions, the largest natural global methane source, may be partly responsible for this rise. The scarcity of in situ atmospheric methane observations in tropical regions may be one source of large disparities between top-down and bottom-up estimates. Here we present 590 lower-troposphere vertical profiles of methane concentration from four sites across Amazonia between 2010 and 2018. We find that Amazonia emits 46.2 ± 10.3 Tg of methane per year (~8% of global emissions) with no temporal trend. Based on carbon monoxide, 17% of the sources are from biomass burning with the remainder (83%) attributable mainly to wetlands. Northwest-central Amazon emissions are nearly aseasonal, consistent with weak precipitation seasonality, while southern emissions are strongly seasonal linked to soil water seasonality. We also find a distinct east-west contrast with large fluxes in the northeast, the cause of which is currently unclear.