Biosensor measurement of purine release from cerebellar cultures and slices

We have previously described an action-potential and Ca2+-dependent form of adenosine release in the molecular layer of cerebellar slices. The most likely source of the adenosine is the parallel fibres, the axons of granule cells. Using microelectrode biosensors, we have therefore investigated whether cultured granule cells (from postnatal day 7–8 rats) can release adenosine. Although no purine release could be detected in response to focal electrical stimulation, purine (adenosine, inosine or hypoxanthine) release occurred in response to an increase in extracellular K+ concentration from 3 to 25 mM coupled with addition of 1 mM glutamate. The mechanism of purine release was transport from the cytoplasm via an ENT transporter. This process did not require action-potential firing but was Ca2+dependent. The major purine released was not adenosine, but was either inosine or hypoxanthine. In order for inosine/hypoxanthine release to occur, cultures had to contain both granule cells and glial cells; neither cellular component was sufficient alone. Using the same stimulus in cerebellar slices (postnatal day 7–25), it was possible to release purines. The release however was not blocked by ENT blockers and there was a shift in the Ca2+ dependence during development. This data from cultures and slices further illustrates the complexities of purine release, which is dependent on cellular composition and developmental stage

A cell-based assay for CD63-containing extracellular vesicles

Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.</div

Fast recharge circuit for q-switched lasers

Cavity-dumped lasers employ electrooptic-effect cell to alternately block and release laser pulse. Cell requires high-speed switching circuit that can apply and remove high voltage. Solid-state circuit employs complementary transistor switches which can switch at rates greater than 5 kHz, eliminate warmup time, provide variable voltage wave-form, and allow polarity reversal

A Versatile Ligand Platform that Supports Lewis Acid Promoted Migratory Insertion

A helping hand: Incorporation of Group 2 Lewis acids into a macrocycle appended to a phosphine ligand attached to a rhenium carbonyl complex promotes otherwise unfavorable transformations of coordinated CO (see scheme; M=Ca, Sr). These Lewis acids form relatively weak M-O bonds, thereby enabling release of organic products from the metal center

Development of Mucoadhesive Gel Microbicide to Target Mucosal HIV Reservoirs

The wide use of microbicide is mainly depends on its effectiveness, less frequent application, ready availability and most importantly cost. The aim of this work was to develop affordable microbicide mucoadhesive gel formulation of synthetic anti HIV drug, stavudine and to characterise it in terms of its physical properties, mucoadhesiveness and spreadability. The purpose of the present study was also to compare different dissolution media used for in vitro release of vaginal dosage form. The gels were tested for antimicrobial, spermicidal and anti-HIV activity. Gels prepared using Carbopols and Polycarbophil were transparent and homogenous and had excellent mucoadhesion index - and showed fast drug release profile. Gels showed very good antimicrobial action against pathological microorganism

Transferable Output ASCII Data (TOAD) gateway: Version 1.0 user's guide

The Transferable Output ASCII Data (TOAD) Gateway, release 1.0 is described. This is a software tool for converting tabular data from one format into another via the TOAD format. This initial release of the Gateway allows free data interchange among the following file formats: TOAD; Standard Interface File (SIF); Program to Optimize Simulated Trajectories (POST) input; Comma Separated Value (TSV); and a general free-form file format. As required, additional formats can be accommodated quickly and easily

Determining the solubility and crystal form of clenbuterol in thin films of eudragit NE30D

The solubility of the drug clenbuterol in thin films of surfactant-free Eudragit NE30D has been measured. Light microscopy and differential scanning calorimetry were supplemented by a technique based on measurement of the rate of drug release from the films. The clenbuterol crystals had the form of a fractal, as could be shown by a computer simulation of diffusion-controlled aggregation

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

The intracellular action on exocytosis of botulinim A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe. The release of the neuropeptide vasopressin was measured by radioimmunoassay. In the presence of the reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 μM free calcium. Half maximal inhibition was obtained with 15 nM botulinum A toxin. In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM. The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin. The heavy chain of botulinum A toxin did not affect vasopressin release. However, it prevented the inhibitory effects of the light chain on stimulated exocytosis. It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals. The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain