The effects of activators of G-proteins,mastoparan and compound 48/80, on the G-protein/adenylate cyclase system in cultured melanophores of the black-moor goldfish,Carassius auratus

 To investigate the functions of GTP-binding protein(s) in the melanosome-aggregating response in fish melanophores, the effects of activators of G-proteins, namely, mastoparan and compound 48/80, were examined in cultured melanophores of the balck-moor goldfish, Carassius auratus. Both mastoparan and compound 48/80 induced an approximately 40% increase in the GTP-hydrolyzing activity in the melanophore membranes compared to the basal level. In intact melanophores, these compounds inhibited the effect of 3-isobutyl-1-methylxanthine, which induced the accumulation of intracellular cAMP. Pretreatment of melanophores with pertussis toxin at 1 μg ⋅ ml^-1 for 15 h attenuated the inhibitory effect of mastoparan on the accumulation of cAMP. However, pretreatment with the toxin only slightly attenuated the inhibitory effect of compound 48/80 on the accumulation of cAMP. In addition, compound 48/80 at 1 mg ⋅ ml^-1 induced full aggregation of the melanosomes in melanophores, though mastoparan at 5 μmol ⋅ l^-1 induced only 10–20% aggregation of melanophores. These results suggest that mastoparan and compound 48/80 can each activate the inhibitory G-protein in goldfish melanophores, which results in inhibition of adenylate cyclase activity. This signal-transduction pathway is involved in the aggregation of melanosomes in these cells. Accepted: 3 June 1996     

Cloning and Characterization of the Murine GPI Anchor Synthesis GenePigf,a Homologue of the HumanPIGFGene

Many eukaryotic proteins are bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Its core backbone, which is conserved in different organisms, is synthesized in the endoplasmic reticulum by the sequential addition of glycan components to phosphatidylinositol. One of the human GPI synthesis genes,PIGF(phosphatidylinositol glycan complementation class F), which is involved late in the synthesis pathway, has been cloned. In this study, we isolated complementary and genomic clones ofPigf,a murine counterpart ofPIGF. Pigfencodes a 219 amino acid protein that complements a class F mutation. ThePigfgene consists of six exons spanning 30 kb and was mapped to chromosome 17 at 17E4–E5. These features are very similar toPIGF,thus demonstrating the interspecies conservation of structure, function, gene organization, and genetic locus between these GPI synthesis genes. The results also extend a region in murine distal chromosome 17 that is syntenic to human chromosome 2p16–p22.     

Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization.

Phenotype and commitment of thymus-colonizing precursors are unknown. Here we report the identification of T lineage-committed precursors (designated prothymocytes) in murine fetal blood at day 15.5 of development. Fetal blood pro-thymocytes are Thy-1+c-kit(low)CD3- in contrast to fetal blood-derived pluripotent hematopoietic progenitors which are Thy-1-c-kit+. Upon transfer into the thymus, fetal blood pro-thymocytes generate a single wave of CD4+CD8+ thymocytes and subsequently mature TCR alpha beta+ peripheral T cells. However, fetal blood pro-thymocytes lack multipotent progenitor potential since they fail to reconstitute B lymphocytes and myeloid and erythroid lineages. In contrast, T and B lymphocytes as well as myeloid and erythroid lineages are reconstituted from fetal blood-derived pluripotent progenitors. Pro-thymocytes are equally present in peripheral blood of athymic fetal mice, suggesting that this novel precursor population is T lineage-committed prior to thymus colonization and represents the earliest T lineage precursor identified.     

Hyperpolarization-activated inward chloride current in protoplasts from suspension-cultured carrot cells

Summary Plasmalemmal ionic currents from enzymatically-isolated protoplasts of suspension-cultured carrot cells were investigated by patch-clamp techniques. Among other currents, a novel hyperpolarization-activated, inwardly-rectifying, whole-cell current was observed. The activation of this current was fast in onset, and for large hyperpolarizations a characteristic, rapid voltage-dependent inactivation was seen. Ion substitution experiments indicate that this inward current was due mainly to efflux of chloride ions. No dependence on either internal or external calcium was found, and internal MgATP was not necessary. Surprisingly, zinc did not block this current. In hyperpolarized outside-out patches, inward single-channel chloride currents having an elementary conductance of ca. 100 pS were observed. The open probability increased with hyperpolarization. Similar single-channel currents were activated by slight negative pressure applied to the pipette. These chloride currents could contribute both to the control of membrane potential and in the regulation of osmotic balance in carrot cells.Abbreviations BAPTA 1,2-bis (2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - E_x Nernst equilibrium potential for ion x - NMDG N-methyl-D-glucamine - PMSF phenylmethylsulfonyl fluoride     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Analysis of effector T cells against the murine syngeneic tumor MethA in mice orally administered antitumor polysaccharide SPR-901

The growth of MethA tumor was significantly inhibited by oral administration of the -glucan SPR-901 in BALB/c (+/+) mice but not in nude mice. Mice treated orally with SPR-901 exhibited an augmentation of antigen-specific resistance against rechallenge with the tumor cells. The tumor-neutralizing activity of regional lymph node cells from MethA-bearing mice against the tumor was augmented by oral administration of SPR-901. The tumor-neutralizing activity of lymph node cells from SPR-901-treated mice mainly appeared in Lyt2+cells. Furthermore, lymphokine-activated killer activity of these cells was enhanced by administration of SPR-901. The antitumor effect of SPR-901 was abrogated in mice depleted of either L3T4+ or Lyt2+ cells, and in cyclosporin-A-treated mice. These results suggest that Lyt2+ cells are important effector cells in MethA-bearing mice orally adminstered SPR-901 and that functional exertion of both Lyt2+ and L3T4+T cells is necessary for the antitumor effect of orally administered SPR-901 in vivo.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

The Presequence of a Precursor to the {delta}-Subunit of Sweet Potato Mitochondrial F1ATPase is not Sufficient for the Transport of {beta}-Glucuronidase (GUS) into Mitochondria of Tobacco, Rice and Yeast Cells

A precursor to the