An 18K protein from ascites hepatoma cell depolymerizes actin filaments rapidly

Several actin binding proteins were isolated from ascites hepatoma cells AH7974 by DNase I affinity chromatography. Among them, a protein having a molecular weight of 18,000 was further purified by DEAE cellulose and hydroxyapatite column chromatographies and gel filtration on a Sephadex G-75 column. The 18K protein not only inhibits actin polymerization but also depolymerizes actin filaments. This conclusion was supported by viscosity and fluorescence intensity measurements and the DNase I inhibition assay. A chemical cross-linking experiment suggested that the 18K protein binds to monomeric actin and forms and 18K-actin 1:1 complex. The net depolymerization rate by the 18K protein measured by the DNase I inhibition assay was slower than the rapid reduction of the fluorescence intensity of pyrene-labeled F-actin upon addition of the 18K protein. This result suggests that the 18K protein not only binds to monomeric actin but also binds to actin filaments directly. The sedimentation assay showed that a part of the 18K protein was cosedimented with actin filaments. Electron microscopic observations demonstrated that the 18K protein decreased the amount of actin filaments and the remaining filaments appeared to be decorated and distorted by the 18K protein. The 18K protein had no Ca2+ ion sensitivity and exhibited the same effect on both this tumor actin and muscle actin.     

Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA

The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. Despite their different pathogenicity, the nucleotide sequences of the env gene of Friend MCF virus and Moloney MCF virus were quite homologous, suggesting that the putative parent sequence for the generation of both MCF viruses and the recombinational mechanism for their generation might be the same. We compare the amino acid sequences in lymphoid leukemia-inducing ecotropic Moloney virus and Moloney MCF virus, and erythroblastic leukemia-inducing ecotropic Friend virus, Friend-MCF virus, and Friend spleen focus-forming virus. The Friend MCF virus long terminal repeat was found to be 550 base pairs long. This contained two copies of the 39-base-pair tandem repeat, whereas the spleen focus-forming virus genome contained a single copy of the same sequence.     

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

Role of hypothalamic histaminergic systems in the regulation of vigilance states in cats

We have studied the effects of local injections of histaminergic and antihistaminic drugs on the sleep-waking cycle in the cat. Microinjections of alpha-fluoromethylhistidine (alpha-FMH), a specific inhibitor of histidine decarboxylase, in the ventrolateral posterior hypothalamus, where histamine-immunoreactive neurons have been recently identified, resulted in a significant decrease in wakefulness (W) and increase in deep slow wave sleep (SWS). On the other hand, microinjections of SKF-91488 (Homodimaprit), a specific inhibitor of histamine-N-methyltransferase, increased W and decreased SWS and paradoxical sleep (PS). Microinjections of histamine also produced an increase of W, while this effect was abolished by pretreatment with mepyramine, an H1-histamine receptor antagonist.     

Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation

Flagellar motility of Triton models of sea urchin spermatozoa was reactivated by cyclic AMP-dependent protein kinase and a protein factor, termed motility activator, both of which were prepared from the detergent-extract of sea urchin spermatozoa. It was shown that phosphorylation of the motility activator by the protein kinase is necessary for the reactivation of flagellar motility [Ishiguro et al, J. Cell Biol. 92:777-782, 1982; Murofushi et al, in "Biological Functions of Microtubules and Related Structures," Academic Press, 1982]. Reactivating factor was also detected in a KCl-extract of the axoneme fraction devoid of the detergent-extractable materials. The activity of this factor was also cyclic AMP- and protein kinase-dependent. Furthermore, when freshly prepared Triton models were treated with phosphoprotein phosphatase prepared from bovine cardiac muscle, the flagellar motility was drastically suppressed. This inhibition of the motility was partially recovered by the addition of cyclic AMP and protein kinase to the phosphatase-treated models.     

Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [³H]γ-aminobutyric acid (pH]GABA) and l -[³H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [³H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l -[³H]glutamate was little affected by taurine. The release of [³H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [³H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [³H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca²⁺ into cerebellar tissues was tentatively suggested.     

Differential staining of insect neurons with nickel and cobalt

Pairs of neurons were differentially stained by intracellular injection of the divalent cations, nickel and cobalt. Pairs of neuronal tracts could be similarly labelled following immersion of their axon bundles in the markers described. Use of nickel and a 4 : 6 mixture of cobalt and nickel according to the protocols presented here revealed the morphology of cricket neuron processes as thin as 1 μm in diameter and yielded a favourable contrast between the cells marked, as well as with respect to the background of unstained tissue.