Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75 % of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90 % of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the £j values for RCAI binding, internalization and efflux were 5, 18 and 13-14 min, respectively. By comparing efflux with and without galactose present, we found that 60 % of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4°C became localized within more caveolae within 2.5 min of warming to 37 °C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN

Using the Product Impact Tool for Prospective Thinking

The ever rising role of products and technologies in humans’ lives is increasing the call for ways to understand and investigate their influences, in the form of prospective analytical methods. This paper proposes one such method, based upon the Product Impact Tool. This Tool was developed to combine both philosophy of technology and design for usability perspectives. Its effects offer potential for prospective and reflective purposes, and can be used to investigate and structure ideas about the impacts of both current and future technologies. The proposed method offers an addition to existing tools within the field of prospective analysis. This added value is demonstrated through a case study of a concept for future personal transport. Through this case study, it is shown that the proposed method can help uncover information that remained hidden by conventional approaches, by inducing a critical investigation of the subject from multiple perspectives. Such information will aid analysts and strategists in their work, leading to more effective, desirable, and responsible technologies being developed and implemented

A simple integrated single-atom detector

We present a reliable and robust integrated fluorescence detector capable of detecting single atoms. The detector consists of a tapered lensed single-mode fiber for precise delivery of excitation light and a multimode fiber to collect the fluorescence. Both are mounted in lithographically defined SU-8 holding structures on an atom chip. Rb87 atoms propagating freely in a magnetic guide are detected with an efficiency of up to 66%, and a signal-to-noise ratio in excess of 100 is obtained for short integration times.Comment: 3 pages, 3 figure

Increased lipophilicity and subsequent cell partitioning decrease passive transcellular diffusion of novel, highly lipophilic antioxidants

ABSTRACT Oxidative stress is considered a cause or propagator of acute and chronic disorders of the central nervous system. Novel 2,4-diamino-pyrrolo [2,3-d]pyrimidines are potent inhibitors of iron-dependent lipid peroxidation, are cytoprotective in cell culture models of oxidative injury, and are neuroprotective in brain injury and ischemia models. The selection of lead candidates from this series required that they reach target cells deep within brain tissue in efficacious amounts after oral dosing. A homologous series of 26 highly lipophilic pyrrolopyrimidines was examined using cultured cell monolayers to understand the structure-permeability relationship and to use this information to predict brain penetration and residence time. Pyrrolopyrimidines were shown to be a more permeable structural class of membrane-interactive antioxidants where transepithelial permeability was inversely related to lipophilicity or to cell partitioning. Pyrrole substitutions influence cell partitioning where bulky hydrophobic groups increased partitioning and decreased permeability and smaller hydrophobic groups and more hydrophilic groups, especially those capable of weak hydrogen bonding, decreased partitioning, and increased permeability. Transmonolayer diffusion for these membrane-interactive antioxidants was limited mostly by desorption from the receiver-side membrane into the buffer. Thus, in this case, these in vitro cell monolayer models do not adequately mimic the in vivo situation by underestimating in vivo bioavailability of highly lipophilic compounds unless acceptors, such as serum proteins, are added to the receiving buffer. A series of novel 2,4-diamino-pyrrolo[2,3-d]pyrimidines were described as potent inhibitors of iron-dependent lipid peroxidation, and proved to be cytoprotective in cell culture models of oxidative injury and neuroprotective in brain injury and ischemia models Structural determinants of permeability and partitioning are discussed for a series of structurally similar homologs. In addition, detailed studies were conducted concurrently with two radiolabled compounds from the pyrrolopyrimidine series representing different physicochemical, permeability, and cell partitioning attributes to discern the roles of protein binding and cell partitioning on permeation and to complement ongoing pharmacological and pharmacokinetic studies. The data proved useful in predicting which compounds were most likely to leave the blood and penetrate underlying tissue. In a companion paper, brain uptake dynamics and cellular penetration of these compounds are confirmed in viv

Advances and Challenges in Protein-Ligand Docking

Molecular docking is a widely-used computational tool for the study of molecular recognition, which aims to predict the binding mode and binding affinity of a complex formed by two or more constituent molecules with known structures. An important type of molecular docking is protein-ligand docking because of its therapeutic applications in modern structure-based drug design. Here, we review the recent advances of protein flexibility, ligand sampling, and scoring functions—the three important aspects in protein-ligand docking. Challenges and possible future directions are discussed in the Conclusion