Human neuroblastoma cell lines having smooth muscle cell markers]

The neural crest gives rise to a variety of tissues, including peripheral neurons, Schwann cells, melanocytes and ectomesenchymal cells, which include the smooth muscle cells of large arteries. Cell lines derived from neuroblastoma (a neural crest tumor) have at least two distinct morphological cell types, a neuroblastic phenotype (N-type) and an epithelial-like phenotype (S-type) with characteristics of substrate-adhesiveness. We have analyzed 17 human neuroblastoma cell lines using a panel of monoclonal antibodies against cytoskeletal proteins. Three neuroblastoma cell lines (KP-N-SI, KP-N-YN and SMS-KCN) bound an alpha -smooth muscle actin antibody. In addition, one of these cell lines (KP-N-SI) bound anti-desmin monoclonal antibodies as determined by indirect immunofluorescence. A total of eight cloned cell lines were obtained from the above parent cell lines. These were composed of either N- or S-type cells and were confirmed to be the common neuroblastoma origin from each parent cell line by chromosomal analysis. Alpha-smooth muscle actin and desmin were demonstrated in the S-type cloned cells by indirect immunofluorescence, as well as by two-dimensional Western blot analysis. These results were confirmed by Northern blot analysis using a specific probe (pSH alpha SMA-3'UT) to human alpha-smooth muscle actin mRNA. This is the first report of the presence of alpha-smooth muscle actin and desmin in neuroblastoma cell lines. These data show that in addition to giving rise to cells with neural, Schwann cell and melanocyte markers, neuroblastoma can also give rise to the cells expressing smooth muscle cell markers.     

Extrachromosomal Element Delta in DROSOPHILA MELANOGASTER. IX. Induction of Delta-Retaining Chromosome Lines by Mutation and Gene Mapping

[Delta b], symbolized as [delta(b)], is retained by S(b) chromosome lines and transmitted through the females to their progeny. Transmission through the males is not directly demonstrable (Minamori 1969a). [delta r], symbolized as [delta(r)], is retained by S(r) chromosome lines and transmitted biparentally (Minamori 1971). The multiplication of delta is suppressed at low temperature. All descendant lines derived from S(b)-carrying or S(r)-carrying flies in which the presence of delta cannot be demonstrated gradually accumulate their specific delta factors over many generations (Minamori 1969b, 1972). The delta factors and the sensitive chromosomes are inseparably associated. This observation led to the assumption that delta may be a copy of a chromosomal gene or a certain agent integrated into the chromosome (Minamori 1972). This assumption was examined in the present study by experiments designed to induce delta-retaining sensitive chromosomes, and to map the gene(s) responsible for delta-retention and/or for sensitivity to the killing action of delta factor. One sensitive chromosome which retained [delta(b)] (S(b) chromosome) was obtained in the presence of [delta(b)] out of 2492 insensitive chromosomes which retained no delta; in addition one S(b) chromosome was obtained in the presence of [delta(r)] out of 2131 insensitives. The latter finding suggests that S(b) might be induced by a mutation caused by [delta(b)] or [delta(r)], but not by integration of either delta into the chromosome. Four S(b) chromosomes and one sensitive chromosome which retained [delta(r)] (S(r) chromosome) were obtained out of 1970 insensitives when males carrying the chromosome were fed an alkylating mutagen, ethyl methane sulfonate (EMS). The location of delta-retaining genes was examined by crossing-over experiments employing eight S(b) and five S(r) chromosomes. The genes on these chromosomes were found to be located in the same region or near one another. The gene for [delta(b)], symbolized as Da(b), and the gene for [delta(r)], symbolized as Da(r), are assumed to be multiple alleles of a locus at 2-24.9. The sensitivity of the chromosomes was modified appreciably by recombination; hence, the genes controlling this trait are assumed to be a polygenic system. The findings obtained in this study lead to the hypothesis that delta may be produced by a chromosomal gene (Da) and transmitted extrachromosomally.     

Synthesis and biological activity of 1alpha-hydroxyvitamin D3

Hydroboration of cholesta-1,5-diene-3β-ol followed by alkaline-peroxide oxidation resulted in the formation of 1α- and 2α-hydroxy derivatives of cholesterol in nearly equal amounts. 1α-Hydroxycholesterol was then transformed to 1α-hydroxyvitamin D₃, via 1α-hydroxycholest-5,7-diene-3β-ol. 1α-Hydroxyvitamin D₃ was as active as 25-hydroxyvitamin D₃ in the stimulation of intestinal calcium transport and bone mineral mobilization in intact rats, and moreover was able to produce both response in anephric rats similar to 1α,25-dihydroxyvitamin D₃, the active metabolite of vitamin D₃, as reported originally by DeLuca's group.     

Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells.

The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway.     

Clostridium perfringens type A enterotoxin induces release of noradrenaline from the neurosecretory PC12 cell line

Clostridium perfringens type A enterotoxin(500 ng/ml) induced extensive release of noradrenaline (1/3-2/3 of the total cell content) from PC12 cells in 2-4 min in the presence, but not the absence of extracellular Ca2+. Cells treated with toxin in the absence of Ca2+ released noradrenaline promptly on subsequent addition of Ca2+ to the medium. The amount of noradrenaline released depended on the concentrations of both Ca2+ and toxin in the medium (ED50, 0.3 mM and 420 ng/ml respectively). Ca2+ could be replaced by Ba2+ or Sr2+, and Mn2+ or Co2+, which are Ca2+ channel blockers, did not inhibit the release of the transmitter. These findings are discussed in relation to the systemic effects of enterotoxin.     

Total synthesis of sialosylcerebroside, GM4

Described are total syntheses of O-[sodium (5-acetamido-3,5-dideoxy-D -glycero-alpha-D-galacto-2-nonulopyranosyl)onate]-(2----3)-O -beta-D -galactopyranosyl-(1----1)-(2R,3S,4E)-2-N-tetracosanoylsphingen ine,O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl+ ++)onate] -(2----3)-O-alpha-D-galactopyranosyl-(1----1)-(2R,3S,4E)-2-N -tetracosanoylsphingenine, O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-beta -D-galacto-2-nonulopyranosyl)onate]-(2----3)-O-beta-D-gal act opyranosyl -(1----1)-(2R,3S,4E)-2-N-tetracosanoylsphingenine, and O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-2-nonulopyranosyl++ +)onate] -(2----3)-O-alpha-D-galactopyranosyl-(1----1)-(2R,3S,4E)-2-N -tetracosanoylsphingenine by using O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D -galacto-2-nonulopyranosyl)onate]-(2----3)-2,3,4,6-tetra-O-a cetyl-D -galactopyrano-syl trichloroacetimidate and O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-beta -D-galacto-2-nonulopyranosyl)onate]-(2----3)-2,4,6-tri-O-ace tyl-D-galactopyranosyl trichloroacetimidate as key glycosyl donors, and (2S,3R,4E)-3 -O-benzoyl-2-N-tetracosanoylsphingenine as a key glycosyl acceptor.     

Interaction between glucocorticoids, 8-bromoadenosine 3',5'-monophosphate, and insulin in regulation of synthesis of carbamoyl-phosphate synthetase I in Reuber hepatoma H-35

Regulation of synthesis of carbamoyl-phosphate synthetase I by glucocorticoids, 8-bromoadenosine 3',5'-monophosphate (8-bromo-cAMP), and insulin was investigated in Reuber hepatoma H-35. By measuring the incorporation of [35S]methionine into carbamoyl-phosphate synthetase I and its precursor, we showed that dexamethasone stimulates the enzyme synthesis approximately fivefold. A detectable stimulation was observed at 1 nM of dexamethasone, half-maximal stimulation at 4 nM, and maximal stimulation above 40 nM. Corticosterone was more effective than dexamethasone both for the minimal concentration needed and for the extent of the stimulation. Hydrocortisone was less effective than dexamethasone. 8-Bromo-cAMP also stimulated the enzyme synthesis at a concentration of 3 mM. The effect of 8-bromo-cAMP was suggested to be additive to the effect of dexamethasone. Physiological concentrations of insulin strongly suppressed the stimulatory effect of dexamethasone on the enzyme synthesis but could not completely counteract the effect of dexamethasone. The half-maximal and maximal effects of insulin were observed at 0.5 nM and 5 nM, respectively. Insulin also counteracted the effect of 8-bromo-cAMP on the enzyme synthesis.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.