Ganglioside Modulation of Cyclic AMP-Dependent Protein Kinase and Cyclic Nucleotide Phosphodiesterase In Vitro

The expression of glial fibrillary acidic protein (GFAP)-mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern-blot evaluation using a specific cDNA probe, and cell-free translation associated with immunoprecipitation. During brain maturation (4-56 days postnatal), the GFAP-mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7-32 days in culture), the GFAP-mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7-18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7-32 days), alpha-tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP-mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.     

Genetic diversity among simian immunodeficiency virus isolates from African green monkeys

To characterize isolates further within the SIVagm subtype, we studied four SIVagm isolates by cross-hybridization, molecular cloning, and nucleotide sequencing. Our results indicate an unexpected degree of genetic variation among isolates within the SIVagm subtype comparable to the variation between SIVmac and HIV-2.     

Relationship between arachidonate generation and exocytosis in permeabilized mast cells.

Using rat mast cells permeabilized with streptolysin O we show that release of arachidonate generally occurs under similar but not identical conditions to those that cause exocytosis of beta-N-acetylglucosaminidase (hexosaminidase). Thus, hexosaminidase secretion and arachidonate release both require provision of Ca2+ together with a guanine nucleotide but exocytosis occurs at lower concentrations of both effectors. The kinetics of both processes are similar, with a delay in onset only when ATP is present. Arachidonate release occurs largely from a pool of arachidonyl phosphatidylcholine which appears to represent less than 1% of the total phosphatidylcholine of the cells. Despite the general similarity of the conditions causing exocytosis and arachidonate release, our results show that under some circumstances it is possible to obtain exocytosis without measurable release of arachidonate and that therefore phospholipase A2 activation is not an essential precursor of secretion.     

Changes in phylloquinone epoxidase activity related to prothrombin synthesis and microsomal clotting activity in the rat

The oxidation of phylloquinone to the 2,3-epoxide (by phylloquinone epoxidase) was studied in liver from control and warfarin-resistant rats. The reaction requires microsomal fraction, soluble protein, a heat-stable soluble factor and O(2). It is not inhibited by CO or CN(-). Epoxidase activity was stimulated if plasma prothrombin was lowered either by anticoagulants or the absence of vitamin K. The activity of the enzyme rapidly returned to normal values after the administration of vitamin K to hypoprothrombinaemic rats. These differences in the activity of the enzyme occur in the microsomal fraction and not the cytosol. A thrombin-generating polypeptide that accumulates in microsomal fraction of hypothrombinaemic rats correlated directly with epoxidase activity. These data support the view that enzymic interconversion of phylloquinone and its 2,3-epoxide participates in the biological activity of vitamin K.     

DEVELOPMENTAL CORRELATES OF GENOME SIZE IN PLETHODONTID SALAMANDERS AND THEIR IMPLICATIONS FOR GENOME EVOLUTION

We present an analysis of the evolutionary relationship between genome size (C-value, mass of DNA per haploid nucleus) and developmental rate using observations of limb regeneration in salamanders of the family Plethodontidae. Rates of growth and differentiation of regenerating limbs are reported for 27 plethodontid species whose C-values range from 14 to 76 picograms. A phylogenetic analysis employing Felsenstein's method of independent contrasts indicates that rate of differentiation is inversely proportional to genome size, although we have not identified any statistically significant association between genome size and the growth rate of regenerating tissue. Our results are consistent with an interpretation that genome size may place a limit on the maximum rate of regeneration attainable in plethodontid salamanders. The implications of our findings for the “junk DNA,” “nucleotypic DNA,” “selfish DNA,” and “skeletal DNA” hypotheses of genome evolution are discussed.     

Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution

Two-affinity purified tetanotoxin forms, TeToA and TeToB, with different affinities for gangliosides were characterized by analytical ultracentrifuge, circular dichroism (CD), and amino acid composition. Both toxin forms share a common sedimentation coefficient of about 6-7 S and similar alpha-helicity values, but they vary in amino acid composition. Incubation of TeToB with micellar polysialogangliosides results in formation of high (21-24 S) and medium (13-15 S) size toxin-micellar ganglioside aggregates as revealed by analytical ultracentrifuge technique. At TeToB/[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GT1b) molar ratios of greater than 26, high molecular weight aggregates (Mr greater than or equal to 700,000) which contain between 3 and 5 toxin monomers are formed, whereas at molar ratios less than 15, about 1-2 monomers are present. TeToA does not form aggregates in the presence of gangliosides. A marked increase in the alpha-helix from about 20 to 39% is apparent in the CD spectrum of TeToB after interaction with ganglioside mixture (G1b). Cerebrosides, sulfatides, sphingomyelin, and phosphatidylserine also increase the alpha-helix, presumably because of an overall effect of lipids on the protein. TeToA and fragment B but not C also undergo similar changes in the presence of G1b, suggesting that the effect of ganglioside is not specific. The polarity of the CD spectra of a number of gangliosides is shifted from a negative to a positive value after interaction with tetanotoxin. The data are consistent with the interpretation of a discrete hydrophobic domain on the toxin heavy chain which interacts with micellar gangliosides to form macromolecular complexes.     

D2 Dopamine Receptors on Bovine Chromaffin Cell Membranes: Identification and Characterization by [3H]N-Methylspiperone Binding

Although dopamine-containing cells are known to be present in sympathetic ganglia, the site of action and the role of dopamine in ganglion function remain obscure. In the present work, we evaluated the interaction of dopamine receptor ligands with particulate membrane fractions from bovine chromaffin cells and adrenal medullary homogenates using the D2 dopamine receptor radioligand [3H]N-methylspiperone ([3H]NMSP). Scatchard analysis of [3H]NMSP saturation experiments revealed a Bmax of 24.1 +/- 1.6 fmol/mg of protein and a KD of 0.23 +/- 0.03 nM in the particulate fraction from adrenal medulla homogenates and a Bmax of 26.5 +/- 2.7 fmol/mg of membrane protein and a KD of 0.25 +/- 0.02 nM in the particulate fraction prepared from isolated adrenal chromaffin cells. There were approximately 1,000 receptors/cell. There were no detectable levels of specific [3H]NMSP binding in the particulates prepared from adrenal cortical or capsular homogenates. Competition studies with the nonradioactive D2 receptor antagonists spiperone, chlorpromazine, and (-)-sulpiride revealed KI values of 0.28, 21, and 196 nM, respectively. The (+) isomer of butaclamol displayed a 604-fold higher affinity than the (-) isomer. Competition studies with the dopamine receptor agonists dopamine and apomorphine revealed affinities of 3,960 and 417 nM, respectively. A correlation coefficient of 0.96 was obtained in studies comparing the potencies of drugs in inhibiting specific [3H]NMSP binding in bovine adrenal medullary homogenates and in inhibiting specific [3H]NMSP binding to brain D2 dopamine receptors. In summary, radiolabeling studies using [3H]NMSP have revealed the presence of D2 dopamine receptors on bovine adrenal chromaffin cells.(ABSTRACT TRUNCATED AT 250 WORDS)