Quantitative determination of the biological activity of botulinum toxin type A by measuring the compound muscle action potential (CMAP) in rats

Quantitative determination of the biological activity of botulinum toxin type A usually depends on the LD₅₀ method after intraperitoneal injection into mice. This method requires a large number of mice to determine the toxic activity at a high level of precision and 3–4 days to obtain the results. Techniques to replace the LD₅₀ method have been attempted at various institutes. As a substitute for this method, by directly measuring the inhibition of neuromuscular transmission after the administration of a toxin, a method to quantitatively assess the toxin's activity by determining the compound muscle action potential (CMAP) was examined.Toxin solutions were injected into the rat gastrocnemius muscle, and that of the CMAP amplitude was determined over time. The CMAP amplitude decreased over 4 days after the injection of the toxin, and then slowly recovered. A dose-response relationship was noted for each dose, and a linear relation was observed between 0.01 and 30 U on the 1st day. From these results, we propose the CMAP as a substitute for the LD₅₀ method to examine the activity of toxin products as it is simple and reliable, reduces the number of experimental animals required, and lowers pain levels.     

Senescence marker protein 30 is up‐regulated in kainate‐induced hippocampal damage through ERK‐mediated astrocytosis

Senescence maker protein 30 (SMP30) is decreased in an androgen‐independent manner in kidney and liver with age. However, regulation of SMP30 expression in the brain has not been examined in aging and neurodegenerative diseases. To investigate SMP30 expression in the brain, we utilized aging and kainate (KA)‐induced neurodegenerative disease models. Interestingly, expression of SMP30 was unlikely to decrease in the aged brain, but total levels of SMP30 protein were increased at 4 weeks after KA injury. Increased glial fibrillary acidic protein (GFAP) with elevated SMP30 expression was observed at the same time post‐KA, indicating that regulation of SMP30 expression in the brain may be associated with astrocytosis. We confirmed that KA induced GFAP expression with increased SMP30 in rat astrocyte cells. Moreover, we found that ERK1/2 activation was involved in the up‐regulation of SMP30 in astrocytes. Our results suggest that elevated SMP30 in activated astrocytes plays an important supportive role after brain damage. © 2009 Wiley‐Liss, Inc.     

Spirostanol pentaglycosides from the underground parts of polianthestuberosa

Phytochemical analysis of the underground parts of Polianthes tuberosa has resulted in the isolation of four new spirostanol saponins with five monosaccharides (1-4). Their structures were determined by spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of hydrolytic cleavage. The cytotoxic activities of 1-4 against HL-60 human promyelocytic leukemia cells and HSC-2 human oral squamous cell carcinoma cells are reported.     

Differential development of cation-chloride cotransporters and Cl- homeostasis contributes to differential GABAergic actions between developing rat visual cortex and dorsal lateral geniculate nucleus

A recent study suggested that gamma-aminobutyric acid (GABA) plays differential roles in activity-dependent plasticity between the visual cortex (VC) and the dorsal lateral geniculate nucleus (dLGN). In the present study, to investigate differential GABAergic functions in postnatal visual system development, the development of [Cl(-)](i), cation-Cl(-) cotransporter expression, and the [Ca(2+)](i) responses evoked by GABA were compared between VC and dLGN during the early stages of development. Using rat brain slices from postnatal days (P) 0-17, GABA-evoked [Ca(2+)](i) responses and resting [Cl(-)](i) were measured by means of optical imaging of Ca(2+) and Cl(-), respectively. Changes in the expression of cation-Cl(-) cotransporters (viz. the outwardly-directed K(+)-Cl(-) cotransporter, KCC2, and the inwardly-directed Na(+),K(+)-2Cl(-) cotransporter, NKCC1) were examined in VC and dLGN by in situ hybridization. At birth, the excitatory actions of GABA were powerful in VC, but missing in dLGN (as indicated by neuronal [Ca(2+)](i) transients), and the resting [Cl(-)](i) was significantly higher in VC than in dLGN. Signals for KCC2 mRNA expression were significantly higher in dLGN than in VC at P0. This suggests that extrusion of Cl(-) from neurons is stronger in dLGN than in VC at P0, so that a GABAergic excitatory effect was not observed in dLGN because of more negative equilibrium potential for Cl(-). The present study indicates clear differences in the molecular and physiological bases of Cl(-) homeostasis and GABA actions between the developing VC and dLGN. Such differential GABAergic actions may underlie the distinct mechanisms involved in VC and dLGN development within the visual system.