Surfactant metabolism and anti-oxidative capacity in hyperoxic neonatal rat lungs: effects of keratinocyte growth factor on gene expression in vivo

Development of preterm infant lungs is frequently impaired resulting in bronchopulmoary dysplasia (BPD). BPD results from interruption of physiologic anabolic intrauterine conditions, the inflammatory basis and therapeutic consequences of premature delivery, including increased oxygen supply for air breathing. The latter requires surfactant, produced by alveolar type II (AT II) cells to lower surface tension at the pulmonary air:liquid interface. Its main components are specific phosphatidylcholine (PC) species including dipalmitoyl-PC, anionic phospholipids and surfactant proteins. Local antioxidative enzymes are essential to cope with the pro-inflammatory side effects of normal alveolar oxygen pressures. However, respiratory insufficiency frequently requires increased oxygen supply. To cope with the injurious effects of hyperoxia to epithelia, recombinant human keratinocyte growth factor (rhKGF) was proposed as a surfactant stimulating, non-catabolic and epithelial-protective therapeutic. The aim of the present study was to examine the qualification of rhKGF to improve expression parameters of lung maturity in newborn rats under hyperoxic conditions (85 % O₂ for 7 days). In response to rhKGF proliferating cell nuclear antigen mRNA, as a feature of stimulated proliferation, was elevated. Similarly, the expressions of ATP-binding cassette protein A3 gene, a differentiation marker of AT II cells and of peroxiredoxin 6, thioredoxin and thioredoxin reductase, three genes involved in oxygen radical protection were increased. Furthermore, mRNA levels of acyl-coA:lysophosphatidylcholine acyltransferase 1, catalyzing dipalmitoyl-PC synthesis by acyl remodeling, and adipose triglyceride lipase, considered as responsible for fatty acid supply for surfactant PC synthesis, were elevated. These results, together with a considerable body of other confirmative evidence, suggest that rhKGF should be developed into a therapeutic option to treat preterm infants at risk for impaired lung development.     

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Background

Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.

Methods

We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.

Results

We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2?>?apoE3?>?apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.

Conclusions

Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

     

Heritabilities,social environment effects and genetic correlations of social behaviours in a cooperatively breeding vertebrate

Social animals interact frequently with conspecifics, and their behaviour is influenced by social context, environmental cues and the behaviours of interaction partners, allowing for adaptive, flexible adjustments to social encounters. This flexibility can be limited by part of the behavioural variation being genetically determined. Furthermore, behaviours can be genetically correlated, potentially constraining independent evolution. Understanding social behaviour thus requires carefully disentangling genetic, environmental, maternal and social sources of variations as well as the correlation structure between behaviours. Here, we assessed heritability, maternal, common environment and social effects of eight social behaviours in Neolamprologus pulcher, a cooperatively breeding cichlid. We bred wild‐caught fish in a paternal half‐sibling design and scored ability to defend a resource against conspecifics, to integrate into a group and the propensity to help defending the group territory (“helping behaviour”). We assessed genetic, social and phenotypic correlations within clusters of behaviours predicted to be functionally related, namely “competition,” “aggression,” “aggression‐sociability,” “integration” and “integration‐help.” Helping behaviour and two affiliative behaviours were heritable, whereas there was little evidence for a genetic basis in all other traits. Phenotypic social effects explained part of the variation in a sociable and a submissive behaviour, but there were no maternal or common environment effects. Genetic and phenotypic correlation within clusters was mostly positive. A group's social environment influenced covariances of social behaviours. Genetic correlations were similar in magnitude but usually exceeding the phenotypic ones, indicating that conclusions about the evolution of social behaviours in this species could be provisionally drawn from phenotypic data in cases where data for genetic analyses are unobtainable.     

Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells: new insights into the pathways involved

Cytokines released by islet-infiltrating immune cells play a crucial role in beta-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this beta-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 +/- 164 (pH 4-7) and 1641 +/- 73 (pH 6-9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p < or = 0.01) by a combination of interleukin-1beta and interferon-gamma, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e-05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a beta-cell dysfunction/apoptotic beta-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to beta-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of beta-cell loss in type 1 diabetes.     

Inundation,Hydrodynamics and Vegetation Influence Carbon Dioxide Concentrations in Amazon Floodplain Lakes

Ecosystems - Extensive floodplains and numerous lakes in the Amazon basin are well suited to examine the role of floodable lands within the context of the sources and processing of carbon within...     

Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.     

NADPH-cytochrome P450 oxidoreductase. Structural basis for hydride and electron transfer

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP(+) has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the re-face of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP(+) lies against the FAD isoalloxazine ring with a tilt of approximately 30 degrees between the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and pi-pi interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.