Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood–brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000? was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.     

Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence

Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called beta-arrestins. Using a knockout mouse lacking beta-arrestin-2 (beta arr2-/-), we have assessed the contribution of desensitization of the mu-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking beta-arrestin-2, desensitization of the mu-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of beta-arrestin-2 does not prevent the chronic morphine-induced up-regulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.     

Inhibition of liver lactate dehydrogenase by serotonin: Possible relationship to endotoxin effects

Zusammenfassung Serotonin hemmt die Lactatdehydrogenase im Lebercytosol von Kaninchen in vitro. Für eine 10%ige Aktivitätshemmung gegenüber den Kontrollwerten genügte eine durchschnittliche Konzentration von 9,15 /ml, die nur etwa zweimal höher liegt als der normale Blutspiegel. Dieser Effekt ist möglicherweise an der im Verhältnis zum Sauerstoffdefizit auffallend geringen Lactatbildung im Endotoxinschock beteiligt.

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Work supported by Research Grant No. 3.10.68 from the Swiss National Foundation for Scientific Research.     

Pregnancy specificβ 1 — a product of the syncytiotrophoblast

Summary Pregnancy specific ₁ (PSG) has been identified in vitro in trophoblast cultures and in vivo, using transmission electron microscopy, in the syncytiotrophoblast, PSG may, like other pregnancy proteins, have immunosuppressive properties.This study was supported by a grant from the Medical Research Council. We thank Dr.D. R. Abramovich for making available the first trimester placentae and MissWanda Emslie for help in the tissue culture studies.     

Kristallisation und Hochreinigung des humanen Plazenta-Laktogens

Summary A purification of human placental lactogen (HPL) by crystallisation is described. The hormone is characterized by the determination of its sedimentation constant, biuret color value and extinction coefficient. The absence of impurities was checked by immunochemical methods using rabbit immune sera to whole human serum as well as to a crude fraction of HPL.     

Science and technology for water purification in the coming decades

One of the most pervasive problems afflicting people throughout the world is inadequate access to clean water and sanitation. Problems with water are expected to grow worse in the coming decades, with water scarcity occurring globally, even in regions currently considered water-rich. Addressing these problems calls out for a tremendous amount of research to be conducted to identify robust new methods of purifying water at lower cost and with less energy, while at the same time minimizing the use of chemicals and impact on the environment. Here we highlight some of the science and technology being developed to improve the disinfection and decontamination of water, as well as efforts to increase water supplies through the safe re-use of wastewater and efficient desalination of sea and brackish water.     

Numerical Investigation of the Influence of Reynolds Number on Probe Measurements

Multi-holepneumaticprobesarecommonlyusedinturbomachinerymeasurementsbecausetheyaresimple,cheap,anddurable,especiallysincetheycansimultaneouslymeasurevelocityandpressure.TheprobemeasurementsarebasedonflowsimilaritywithReasoneofthebasicsimilaritycriteria.However,theinfluenceofReonprobemeasurementsisnotconsideredinprobecalibrationsbecauseofthedifficultiesinherentinRebasedcalibration.MostofthelimitedexperimentalinvestigationsonthisproblemfocusedondeterminingRerangeinwhichtheinfluencecanbeneglect…