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

Overcoming the blood-brain barrier to taxane delivery for brain tumors and neurodegenerative diseases and brain tumors

The original publication is available at www.springerlink.comThe blood-brain barrier (BBB) effectively prevents microtubule stabilizing drugs from readily entering the central nervous system (CNS). A major limiting factor for microtubule stabilizing drug permeation across the BBB is the active efflux back into the circulation by the over-expression of the multidrug resistant gene product (MDR1) or P-glycoprotein (P-gp). This study has focused on strategies to overcome P-gp-mediated efflux of taxol analogues, microtubule (MT) stabilizing agents that could be used to treat brain tumors and, potentially, neurodegenerative diseases such as Alzheimer’s disease. However, taxol is a strong P-gp substrate which limits its distribution across the BBB and therapeutic potential in the CNS. We have found that addition of a succinate group to the C-10 position of taxol results in an agent, Tx-67, with reduced interactions with P-gp and enhanced permeation across the BBB in both in vitro and in situ models. Our studies demonstrate the feasibility of making small chemical modifications to taxol to generate analogues with reduced affinity for the P-gp but retention of MT-stabilizing properties, i.e., a taxane that may reach and treat therapeutic targets in the CNS

Paclitaxel Succinate Analogs: Anionic Introduction as a Strategy to Impart Blood Brain Barrier Permeability

A focused library of TX-67 (C10 hemi-succinate) analogs have been prepared including regioisomeric, functional group, and one-carbon homologs. These were prepared to investigate TX-67’s lack of interaction with P-glycoprotein (Pgp). Tubulin stabilization ability, cytotoxicity, and Pgp interactions were evaluated. All carboxylic acid analogs had no apparent interactions with Pgp whereas the ester variants of the same compounds displayed characteristics of Pgp substrates. Furthermore, it is demonstrated that hydrogen-bonding properties were significant with respect to Pgp interactions. This anionic introduction strategy may allow for delivery of paclitaxel into the CNS as well as establishing a new method for delivery of other, non-CNS permeable drugs

A relevant in vitro rat model for the evaluation of blood-brain barrier translocation of nanoparticles

Poly(MePEG2000cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to reach the rat central nervous system after intravenous injection. For insight into the transport of colloidal systems across the blood-brain barrier (BBB), we developed a relevant in vitro rat BBB model consisting of a coculture of rat brain endothelial cells (RBECs) and rat astrocytes. The RBECs used in our model displayed and retained structural characteristics of brain endothelial cells, such as expression of P-glycoprotein, occludin and ZO-1, and immunofluorescence studies showed the specific localization of occludin and ZO1. The high values of transendothelial electrical resistance and low permeability coefficients of marker molecules demonstrated the functionality of this model. The comparative passage of polyhexadecylcyanoacrylate and PEG-PHDCA nanoparticles through this model was investigated, showing a higher passage of PEGylated nanoparticles, presumably by endocytosis. This result was confirmed by confocal microscopy. Thanks to a good in vitro/in vivo correlation, this rat BBB model will help in understanding the mechanisms of nanoparticle translocation and in designing new types of colloidal carriers as brain delivery systems

Action potentials in abscisic acid-deficient tomato mutant generated spontaneously and evoked by electrical stimulation

Action potentials generated spontaneously (SAPs) and evoked by electrical stimulation (APs) in tomato plants (Solanum lycopersicum L.) cv. Micro-Tom ABA-deficient mutants (sitiens—MTsit) and its wild type (MTwt) were characterized by continuous monitoring of electrical activity for 66 h and by application of an electrical stimulation supplied extracellularly. MTsit generated SAPs which spread along the stem, including petioles and roots with an amplitude of 44.6 ± 4.4 mV, half-time (t½) of 33.1 ± 2.9 s and velocity of 5.4 ± 1.0 cm min−1. Amplitude and velocity were 43 and 108 % higher in MTsit than in MTwt, respectively. The largest number of SAPs was registered in the early morning in both genotypes. MTsit was less responsive to electrical stimuli. The excitation threshold and the refractory period were greater in MTsit than in MTwt. After current application, APs were generated in the MTwt with 21.2 ± 2.4 mV amplitude and propagated with 5.6 ± 0.5 cm min−1 velocity. Lower intensity stimuli did not trigger APs in these plants. In MTsit APs were measured with amplitude of 26.8 ± 4.8 mV and propagated with velocity of 8.5 ± 0.1 cm min−1

Efeitos de retardadores de crescimento na frutificação da videira 'Niagara Rosada'

Studies were carried out to establish the effects of exogenous growth regulators on Vitis (labrusca x vinifera) 'Niagara Rosada' fruiting. The investigations were done in the Jundiaí Research Station, Agronomic Institute State of São Paulo, always using disease-free vineyards of good productivity. The morphological transformations of clusters were studied under the following aspects: weight, length and width of cluster; weight, length average and width average of berries: length average/width average ratio of berries; length and diameter of rachis; width of cluster minus berries; length and diameter of secondary rachis. The yield for the first half of the period from flowering to maturation was first determined. The same characteristics were determined at the time of maturity plus the number of berries, number of seeds, total sugars, total acid, Maturity Index and reducing sugars in samples of all treatments. The experiment was conducted in order to determine the doses that resulted in the most beneficial effects, always using applications by immersion of the inflorescence. In the experiment was realized applications of (2-chloroethyl) trimethylammonium chloride (CCC) and succinic aeid-2, 2-dimethylhydrazide (SADH) at concentrations of 50, 100, 250, 500, 1000 and 2000 ppm; CCC 500 ppm plus SADH 500 ppm and nontreated, 5 days before flowering, in 1971. The concentrations of CCC applied before flowering did not affect favorably cluster morphology under the conditions of the experiment. Application of SADH at 250 ppm before flowering increased the cluster weight and length, berries number and weight, and seed number. In the first yield treatment of 1000 ppm of SADH increased the cluster weight and lenght, berry weight and rachis lenght.Estudou-se a influência da aplicação por imersão, de retardadores de crescimento (CCC e SADH), 5 dias antes do florescimento, nas características morfológicas da panícuia da videira Vitis (labrusca x vinifera) 'Niagara Rosada'. Neste ensaio verificou-se que as concentrações de CCC aplicadas em pré-florescimento, não afetaram favoravelmente a morfologia das panículas da cultivar estudada, nas condições do ensaio. SADH na dosagem de 1000 ppm provocou, na primeira colheita, aumento no peso e comprimento da panícula, no peso das bagas, e no comprimento da ráquis, proporcionando a formação desejada de uma panícula mais alongada, nas condições estudadas. Aplicação de SADH na concentração de 250 ppm em pré-florescimento, promoveu aumento no peso e comprimento da panícula, número e peso das bagas, além do inconveniente de elevar o número de sementes