Mouse neuroblastoma

Sensitive to toxins, in this case means that the assay presents no false negative results. Primary hepatocytes can elucidate hepatotoxins, and mouse neuroblastoma cells can elucidate sodium channel-blocking neurotoxins therefore these assays can be used to screen for the appropriate toxins. 

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

Tamplin et. al. (54) observed that V. cholerae and A. hydrophila cell extracts contained substances with TTX-like biological activity in tissue culture assay, counteracting the lethal effect of veratridine on ouabain-treated mouse neuroblastoma cells. Concentrations of TTX-like activity ranged from 5 to 100 ng/L of culture when compared to standard TTX. The same bacterial extracts also displaced radiolabelled STX from rat brain membrane sodium channel receptors and inhibited the compound action potential of frog sciatic nerve. However, the same extracts did not show TTX-like blocking events of sodium current when applied to rat sarcolemmal sodium channels in planar lipid bilayers. 

CTx that has been purified from muscles of Gymnothorax javanicus stimulates the release of neurotransmitters such as 7-aminobutyric acid and dopamine from rat brain nerve terminals. It causes a membrane depolarization of mouse neuroblastoma cells and, under appropriate conditions, it creates spontaneous oscillations of... 

Richelson, E. (1978). Histamine HI receptor-mediated guanosine 3, 5 -monophosphate formation by cultured mouse neuroblastoma cells. Science 201, 69-71. 

Holloway, S.F., V.L. Salgado, C.H. Wu, and T. Narahashi. 1989. Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells. Pflugers Archiv. (European Jour. Physiol.) 414 613-621. 

Fredrickson, P. A., and Richelson, E. (1979) Hallucinogens antagonize histamine H, receptors of cultured mouse neuroblastoma cells. Eur. J. Pharmacol., 56 261-264. 

Narahashi T, Tsunoo A, Yoshii M (1987) Characterization of two types of calcium channels in mouse neuroblastoma cells. J Physiol (Lond) 383 231-249... 

In contrast to this work on whole brain, Kemp and Stoolmiller138 used cultured mouse-neuroblastoma cells for their study of incorporation of [ lH]2-acetamido-2-deox> -D-mannose into the sialic acid moiety of gangliosides. They were able to demonstrate clearly the precursor-product relationship among CMS, GM2, and GM1. They found that cultured NB41A cells incorporated [3H]2-acetamido-2-deoxy-D-man-nose into the sialic acid moiety of GM3 in less than 10 minutes. Labeled GM2 was not detected in cells incubated for less than 30 minutes, and measurable activity did not appear in G I1 until alter 60 to 90 minutes. These results further supported the concept that the pathway of synthesis of ganglio-tvpe gangliosides proceeds by way of GM3 —> GM2 — GM1. 

The next channel to be dealt with is the Cx40 channel. The unique conductance and gating of gap junction channels formed by Cx40 has been investigated in Cx40-transfected mouse neuroblastoma cells (N2A cells). In... 

Methyl methanesulfonate (250 iM) induced neurite formation in 71% of mouse neuroblastoma N-18 cells, when cell growth was inhibited by 83% (Yoda et al., 1982). 

WHO (1993) Guidelines for Drinking Water Quality, 2nd Ed., Vol. 1, Recommendations, Geneva Yoda, K., Shimizu, M. Fujimura, S. (1982) Induction of morphological differentiation in cultured mouse neuroblastoma cells by alkylating agents. Carcinogenesis, 3, 1369-1371... 

Lang, D. G., Wang, C. M., Cooper, B. R. Lamotrigine, phenytoin and carbamezepine interactions on the sodium current present in N4TG1 mouse neuroblastoma cells, J. Pharmacol. Exp. Therap. 1993, 266, 829-835. 

Cell Culture. Human neuroblastoma IMR-32 (passaged through nude mice the cells were donated by Dr. Steven E. Brooks, Kingsbrook Jewish Medical Center, Brooklyn) and mouse neuroblastoma clones NIE-115, NS-20, and N-18) (donated by Dr. Shraga Makover, Hoffmann LaRoche, Inc., Nutley, New Jersey) were maintained in our laboratory as described previously (30,31). Confluent monolayers (6 to 8 x 10 cells per 250-ml Falcon plastic flask) were harvested for enzymatic studies with phosphate-buffered saline [7.0 mM potassium phosphate/0.14 M NaCl - buffer, pH 7.2 (Pi/NaCl)] containing 0.1% EDTA. 

In order to obtain some idea about the nature of gly-coconjugates and their gross topographical orientation on the cell surfaces, we measured the binding of 125j labeled lectins and toxin to human neuroblastoma IMR-32 and mouse neuroblastoma N1E-115, NS-20, and N-18 clones (Tables VII and VIII). The "5% TCA Wash" column (Table VII) represents 1251-iabeled lectin or toxin bound to both glycoprotein and glycolipid. The "5%... 

I]Lectin Added Human Neuroblastoma Mouse Neuroblastoma ... 

Figure 3. Effect of chemical differentiating agents on radioactive Bandeiraea simplicifolia [llsI]Iectin binding to mouse neuroblastoma (NlE-115) cell surfaces...

The apparent Km values of the enzyme systems for CMP-NeuNAc assayed with 0.5 mg enzyme protein, was 0.13 mM (same with all four types of acceptors (15)). This value is comparable to that (0.15 mM) obtained (22) in cultured mouse neuroblastoma cells with lactosylceramide as the NeuNAc acceptor. The Km value reported previously (23) for the calf brain enzyme with desialy-lated ai-acid glycoprotein as the acceptor is 4-fold higher. 

Ratio of nitrogen versus phosphate Cell line (Mouse neuroblastoma cells)... 

N1E-115 Mouse neuroblastoma Cholinergic neuron Lead Pyrethroid insecticide Blockage of voltage-dependent Ca2+ channels Prolonged open time for voltage-dependent Na+ channels... 

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