In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

Spider toxin (JSTX) on the glutamate synapse

A new neurotoxin (JSTX) was separated from spider (Nephila clavata, Joro spider) venom. JSTX irreversibly suppressed the excitatory postsynaptic potential (EPSP) and the glutamate potential in the lobster neuromuscular junction with high degree of specificity. The threshold concentration for suppressing EPSPs corresponds to a small fraction of the toxin in a venom gland, roughly estimated as low as 10(-10) M/l. 10(-10) M/l. In the giant synapse of squid stellate ganglion JSTX suppressed EPSPs without affecting the antidromic response. Glutamate-induced membrane depolarization was blocked by JSTX. In mammalian brain slice preparation, JSTX suppressed the orthodromic spike response but failed to affect on the antidromic spike in the hippocampal pyramidal neuron of CA1 and CA3 region. The above results strongly support the view that the squid giant synapse and synapses in the hippocampal pyramidal neuron are mediated by glutamate.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Codon Substitution in Evolution and the "Saturation" of Synonymous Changes

A mathematical model for codon substitution is presented, taking into account unequal mutation rates among different nucleotides and purifying selection. This model is constructed by using a 61 X 61 transition probability matrix for the 61 nonterminating codons. Under this model, a computer simulation is conducted to study the numbers of silent (synonymous) and amino acid-altering (nonsynonymous) nucleotide substitutions when the underlying mutation rates among the four kinds of nucleotides are not equal. It is assumed that the substitution rates are constant over evolutionary time, the codon frequencies being in equilibrium, and, thus, the numbers of synonymous and nonsynonymous substitutions both increase linearly with evolutionary time. It is shown that, when the mutation rates are not equal, the estimate of synonymous substitutions obtained by F. Perler, A. Efstratiadis, P. Lomedico, W. Gilbert, R. Kolodner and J. Dodgson's "Percent Corrected Divergence" method increases nonlinearly, although the true number of synonymous substitutions increases linearly. It is, therefore, possible that the "saturation" of synonymous substitutions observed by Perler et al. is due to the inefficiency of their method to detect all synonymous substitutions.     

Changes in Microtubules in Onion Leaf Sheath Cells during Bulb Development

Cortical microtubules are oriented transversely to the cellaxis in leaf sheath cells of onion plants (Allium cepa L. cv.Osaka-Okute) that have not started bulb formation. As the bulbdevelops and the leaf sheath cells swell, the microtubules becomedisoriented and scattered and finally disappear. The microtubuleinhibitors colchicine and cremart [O-ethyl O-(3-methyl-6-nitrophenyl)N-sec-butylphosphorothioamidate]cause swelling of leaf sheath cells and make the basal partof the plant bulbous. The cortical microtubules may have animportant role in regulating bulb development in onion plants. (Received August 21, 1982; Accepted December 6, 1982)     

Effect of TPA on the mutagenicity of caffeine in the soybean mutation test

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a well-known tumor promoter in mouse-skin carcinogenesis. Its effects on mutagenesis in a soybean test system were examined, and the effects were judged from the appearance of spots of various colors on the leaves. When soybean seeds were treated with TPA plus 0.03% caffeine, the frequency of spots per leaf decreased significantly and in proportion to the concentration of TPA. TPA alone at concentrations of 1–20 μg/ml did not induce any mutations. Mutations induced by γ-rays were not affected by administration of TPA either before or after exposure to γ-rays. The mechanism of suppression by TPA of mutations induced by caffeine is discussed.     

Genotoxicity of quinone pigments from pathogenic fungi

The genotoxicity and mutagenicity of several kinds of quinone pigments from pathogenic fungi were examined by means of the hepatocyte primary culture (HPC)/DNA repair test and of Ames test with TA98 and TA100. Clear genotoxicity of the two quinone chemicals, xanthomegnin and luteosporin were observed in the HPC/DNA repair test, though definite mutagenicity was not detected in the Salmonella microsome test. These two pigments are thus suspected to be genotoxic carcinogens.