EFFECT OF VINBLASTINE AND COLCHICINE ON UPTAKE AND RELEASE OF PUTATIVE TRANSMITTERS BY SYNAPTOSOMES AND ON BRAIN ACTOMYOSIN-LIKE PROTEIN

Abstract— The effects of several inhibitors, including vinblastine and colchicine, on the accumulation of a number of putative transmitters by a rat brain synaptosomal preparation and their subsequent release by excess K⁺ was examined. In addition, the effect of the alkaloids on the ATPase activity of the actomyosin-like protein, neurostenin, isolated from the synaptosomal preparation, was studied. The uptakes of radioactive glutamate, GABA, dopamine and norepinephrine were energy-dependent, as evidenced by their susceptibility to 0.01 mM carbonyl cyanide m-chlorophenylhydrazone (Cl-CCP), 01 mM ouabain and temperature. The active accumulations of GABA, dopamine and norepinephrine were also greatly inhibited by 1 mM6-hydroxydopamine (6-OHDA), 01 mM mersalyl, 0.05–0.25mM vinblastine and 0.1–1.0 mM colchicine. Vinblastine was approximately 10-fold more potent (K₁, ?0.1 mM) than colchicine as an inhibitor. The release of actively accumulated dopamine or norepinephrine by excess K⁺ (increasing the [K⁺] from 5 to 30 mM) was inhibited somewhat when vinblastine was present during the entire incubation period. If the synaptosomes were preloaded with the radioactive compounds prior to addition of vinblastine, there was no discernible effect on the relative amount of material released by excess K⁺. However, the addition of inhibitor under the latter conditions caused a leakage of radioactivity into the medium even without excess K⁺ being present. Glutamate accumulation was somewhat different from that of GABA, dopamine or norepinephrine. Although it required energy for uptake, 6-OHDA, mersalyl, vinblastine or colchicine were not inhibitory. Studies of the oxidative metabolism of glutamate and GABA by this synaptosomal preparation indicated that the mechanisms of inhibition by vinblastine was not attributable to a metabolic effect. Both vinblastine and colchicine inhibited the Mg²⁺-stimulated, but not the Ca²⁺-activated ATPase of neurostenin. This effect was probably attributable to an interaction of the vinblastine with the neurin moiety of this actomyosin-like protein. We suggest that the inhibitory phenomena exhibited by vinblastine and colchicine in this synaptosomal preparation arose from the effect of these alkaloids on the neurin associated with the synaptic membrane.     

Large-Scale Production of Nanographite by Tube-Shear Exfoliation in Water

The number of applications based on graphene, few-layer graphene, and nanographite is rapidly increasing. A large-scale process for production of these materials is critically needed to achieve cost-effective commercial products. Here, we present a novel process to mechanically exfoliate industrial quantities of nanographite from graphite in an aqueous environment with low energy consumption and at controlled shear conditions. This process, based on hydrodynamic tube shearing, produced nanometer-thick and micrometer-wide flakes of nanographite with a production rate exceeding 500 gh^-1 with an energy consumption about 10 Whg^-1. In addition, to facilitate large-area coating, we show that the nanographite can be mixed with nanofibrillated cellulose in the process to form highly conductive, robust and environmentally friendly composites. This composite has a sheet resistance below 1.75 Ω/sq and an electrical resistivity of 1.39×10^-4 Ωm and may find use in several applications, from supercapacitors and batteries to printed electronics and solar cells. A batch of 100 liter was processed in less than 4 hours. The design of the process allow scaling to even larger volumes and the low energy consumption indicates a low-cost process.     

CHROMOSOME MICROMANIPULATION : III. Spindle Fiber Tension and the Reorientation of Mal-Oriented Chromosomes

Kinetochore reorientation is the critical process ensuring normal chromosome distribution. Reorientation has been studied in living grasshopper spermatocytes, in which bivalents with both chromosomes oriented to the same pole (unipolar orientation) occur but are unstable: sooner or later one chromosome reorients, the stable, bipolar orientation results, and normal anaphase segregation to opposite poles follows. One possible source of stability in bipolar orientations is the normal spindle forces toward opposite poles, which slightly stretch the bivalent. This tension is lacking in unipolar orientations because all the chromosomal spindle fibers and spindle forces are directed toward one pole. The possible role of tension has been tested directly by micromanipulation of bivalents in unipolar orientation to artificially create the missing tension. Without exception, such bivalents never reorient before the tension is released; a total time "under tension" of over 5 hr has been accumulated in experiments on eight bivalents in eight cells. In control experiments these same bivalents reoriented from a unipolar orientation within 16 min, on the average, in the absence of tension. Controlled reorientation and chromosome segregation can be explained from the results of these and related experiments.     

Microsatellite markers for the wood decay fungus Phlebiopsis gigantea

Nine polymorphic microsatellite markers were developed for the wood-decay basidiomycete Phlebiopsis gigantea, which is used commercially as a biocontrol agent for annosum root disease on conifers. Microsatellite sequences were isolated from repeat-enriched genomic libraries. Primers flanking these sequences were screened on P. gigantea isolates from Europe and North America. The number of alleles per locus ranged from 5 to 15, and gene diversity ranged from 0.72 to 0.90. These markers should be useful for studies of P. gigantea natural population structure and for making predictions about the impact of P. gigantea application in conifer forests.     

Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes

Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle.     

Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.