5,8-Disubstituted indolizidines: A new class of noncompetitive blockers for nicotinic receptor-channels

A series of 8-methyl-5-substituted indolizidines inhibit binding of the noncompetitive blocking agent [³H]perhydrohistrionicotoxin to muscle-type nicotinic acetylcholine receptor-channels in membranes fromTorpedo electroplax. The K_i values range from 0.16 to 1.12 M, making these alkaloids among the most potent ligands for this site. Unlike most noncompetitive blockers, the potencies of the 8-methyl-5-substituted indolizidines arereduced in the presence of carbamylcholine. Indolizidine 205A (8-methyl-5-(4-pentynyl)indolizidine) is unique in enhancing binding of [³H]perhydrohistrionicotoxin by 1.5-fold. The enhancement is at a maximum at 0.01 to 0.1 M, followed by progressive inhibition with an IC₅₀ of about 20 M. In the presence of carbamylcholine, which itself enhances binding of [³H]perhydrohistrionicotoxin, indolizidine 205A causes only an inhibition of binding with an IC₅₀ of about 10 M. Indolizidines with a hydroxy substituent on the 8-methyl group have very low activity. None of the indolizidines affect binding of [¹²⁵I]-bungarotoxin to acetylcholine recognition sites. In pheochromocytoma PC12 cells, indolizidine 205A has no agonist activity, but only inhibits carbamylcholine-elicited²²Na⁺ influx. The profile of potencies for the 8-methyl-5-substituted indolizidines is similar in electroplax membranes and PC12 cells. Indolizidines 205A and 209B (8-methyl-5-pentylindolizidine) have no apparent effect on desensitization of receptors in PC12 cells. The 5,8-disubstituted indolizidines appear to represent an atypical and potent class of noncompetitive blockers for muscle-type and ganglionic nicotinic receptor-channels.     

Significance of Spermidine in the Initiation of Adventitious Buds in Stem Segments of Torenia

When stem segments of Torenia fournieri Lind. were culturedin vitro, the initiation of adventitious buds, which is usuallyinduced by cytokinin, was induced by application of polyamines,such as putrescine and spermidine. Addition of arginine hada slight inductive effect. When cyclohexylamine, an inhibitorof spermidine synthase, was added simultaneously with putrescine,induction of the initiation of buds by putrescine was stronglyinhibited. However, application of the inhibitor together withspermidine had no effect on the spermidine-induced initiationof buds. The induction of initiation of buds by a cytokinin,by a calcium ionophore, by cyclic AMP, and by a phorbol ester,which was accompanied in each case by elevation of the levelsof endogenous spermidine, was also inhibited by cyclohexylamine.These results suggest the involvement of spermidine in the initiationof adventitious buds in stem segments of Torenia. ²Present address: Radiation Effects Research Foundation, Hijiyamakouen5-2, Minami-ku, Hiroshima, 732 Japan.     

Prediction of Rate‐Limiting Reactions for Growth‐Associated Production Using a Constraint‐Based Approach

Identification of a rate‐limiting step in pathways is a key challenge in metabolic engineering. Although the prediction of rate‐limiting steps using a kinetic model is a powerful approach, there are several technical hurdles for developing a kinetic model. In this study, an in silico screening algorithm of key enzyme for metabolic engineering is developed to identify the possible rate‐limiting reactions for the growth‐coupled target production using a stoichiometric model without any experimental data and kinetic parameters. In this method, for each reaction, an upper‐bound flux constraint is imposed and the target production is predicted by linear programming. When the constraint decreases the target production at the optimal growth state, the reaction is thought to be a possible rate‐limiting step. For validation, this method is applied to the production of succinate or 1,4‐butanediol (1,4‐BDO) in Escherichia coli, in which the experimental engineering for eliminating rate‐limiting steps has been previously reported. In succinate production from glycerol, nine reactions including phosphoenolpyruvate carboxylase are predicted as the rate‐limiting steps. In 1,4‐BDO production from glucose, eight reactions including pyruvate dehydrogenase are predicted as the rate‐limiting steps. These predictions include experimentally identified rate‐limiting steps, which would contribute to metabolic engineering as a practical tool for screening candidates of rate‐limiting reactions.     

Genetically engineered herbicide resistance in cyanobacterium Synechococcus sp. by bialaphos resistance gene from Streptomyces hygroscopicus

A novel cyanobacterial vector, pTT201, containing the bar gene encoding resistance to herbicides, bialaphos and phosphinothricin, was constructed. In Synechococcus sp. strain PCC7942-SPc, the bar gene was successfully expressed. Plasmid pTT201 increased a minimum inhibitory concentration for bialaphos 16-fold over Synechococcus sp. strain PCC7942-SPc without pTT201. The combination of the bialaphos as a selective agent and the transformation by bar gene serves as a photostable selection system for Synechococcus.     

Estimation of seasonal changes in translocation rates in leaves of a Japanese larch stand

To estimate the net rate of translocation in leaves of a larch stand, a new approach based on the summation method was proposed and given by a compartment model. The difference between the rate of translocation into and out of leaf biomass, namely, the net translocation rate (ΔT _r /Δt), was usually expressed by the difference between the growth rate of leaf biomass and the surplus production rate provided that the rate of leaf loss due to leaffall and grazing can be considered negligible. The rate, ΔT _r /Δt in a 19-yr-old larch stand, showed characteristic changes; it was positive from early April to late May, but after that it was negative until leaffall in late October. Our results confirmed that for the growth phase of positive ΔT _r /Δt translocation of assimilate stored in non-photosynthetic organs was indispensable for the growth. To quantify this, the ratio of ΔT _r /Δt to growth rate of leaf biomass was proposed.     

Bovine colostric transforming growth factor-beta-like peptide that induces growth inhibition and changes in morphology of human osteogenic sarcoma cells (MG-63)

TGF-beta like peptide, termed TGF(BC-1), was partially purified from defatted and decaseinated bovine colostrum by a sequence of DEAE-Sephacel chromatography and Sephadex G-50 gel filtration in 1M acetic acid. TGF(BC-1) was distinct from well-known 25K TGF-beta in chemical properties: TGF(BC-1) was sensitive to acid ethanol extraction (Roberts et al., 1980). Its apparent molecular weight ranged from 21k to 11k by gel filtration and it was composed of low MW peptides (15k, 13k, 10k and 7.3k but not 25k) as examined by SDS-PAGE under non-reducing conditions. However, TGF(BC-1) shares some biological properties with the prototype TGF-b. TGF(BC-1) remarkably suppressed growth of osteogenic sarcoma cells (MG-63), and this was intriguingly accompanied by a striking change in morphology.