[IgM Antibody against IgG Antibody Binding to Antigen, So-Called Anti-Antibody, Reacts Mainly with F (ab')2 Fragment Possessing Antibody Activity].

OBJECTIVE: To develop enzyme-linked immunosorbent assay (ELISA) for measurement of IgM antibody (anti-antibody) against human IgG antibody that is binding to antigen, to distinguish anti-antibody from other antiglobulins, e.g. rheumatoid factor (RF), and to search their localization of epitope on IgG molecule. METHODS: We chose purified human IgG anti-dsDNA antibody from a patient with systemic lupus erythematosus as IgG antibody and calf thymus dsDNA as antigen. IgG F (ab')2 fragment was obtained by digestion of the IgG with pepsin. Those IgG and IgG F (ab') 2 with anti-dsDNA antibody were reacted with pre-coated dsDNA and formed immune complex on ELISA plate. On the other hand we prepared ELISA plate on which those IgG and IgG F (ab') 2 were coated directly for measurement of IgM RF and IgM antihinge antibody. Twenty-three sera from patients with rheumatoid arthritis were tested. RESULTS: Correct IgM anti-antibodies were obtained after subtraction of absorbance of IgM anti-dsDNA anti- bodies. Remarkable correlation between IgM anti-antibodies obtained by using whole IgG and those by using IgG F(ab')2(n=23, r²=0.914, p=1.11X10-12). There were correlations neither between IgM antiantibodies and IgM RF(n=23, r²=0.001, p=0.889) nor between IgM antiantibodies and IgM antihinge antibodies (n=23, r²=0.063, p=0.249). CONCLUSIONS: IgM molecule with anti-antibody specificity seems to be different from IgM RF and IgM antihinge antibody. Moreover, epitope recognized by IgM anti-antibody seems to localize on IgG F (ab') 2 but not on hinge region. [Original].

Different effects of α-chloralose on spontaneous and evoked GABA release in rat hippocampal CA1 neurons.

The effects of α-chloralose on presynaptic GABA(A) receptors were investigated with respect to spontaneous and evoked GABAergic transmission (sIPSCs and eIPSCs) in rat hippocampal CA1 pyramidal neurons. sIPSCs were recorded in mechanically dissociated CA1 neurons with intact GABAergic terminals, namely the "synaptic bouton preparation." eIPSCs were elicited by focal electrical stimuli of a single GABAergic bouton on an isolated CA1 neuron using the whole-cell patch recording configurations under voltage-clamp condition. We found that α-chloralose potentiated the exogenous GABA-induced Cl(-) response in a concentration dependent manner, and the drug itself induced Cl(-) response at high concentrations (>100 µM). α-Chloralose at low concentrations (3-10 µM) increased sIPSC frequency without affecting the current amplitude and kinetics, but prolonged the slow current decay time constant (τ(s)) at concentrations greater than 30 µM without changing either current amplitude or frequency. α-Chloralose at 10 µM enhanced amplitude of eIPSCs and decreased the failure rate (Rf), but at 30 µM decreased the amplitude and increased the Rf. Pretreatment with bumetanide, a blocker of NKCC-1, completely prevented the 30 µM α-chloralose-induced inhibition on eIPSC amplitude and Rf. These results suggest that α-chloralose activates GABA(A) receptors on GABAergic presynaptic nerve terminals and depolarizes the terminals, mediating presynaptic inhibition or autoregulation, in a concentration-dependent manner. In addition, α-chloralose at high concentrations activates not only extrasynaptic GABA(A) receptors on the postsynaptic soma membrane but also synaptic GABA(A) receptors resulting in prolongation of current decay phase. Thus α-chloralose induces complex and differential modulation of sIPSCs and eIPSCs in a concentration dependent manner.

Comparative study on the actions of toxin extracts from two different puffer fishes on I(Na) and respiratory N-M transmission in the rat.

We performed a comparative study on the effects of toxin extracts prepared from muscle and liver of two different puffer fishes on voltage dependent sodium current (I(Na)), and compared the results with that of tetrodotoxin (TTX). The amount of toxin contained in the muscle or liver expressed as an amount of equipotent TTX differed in the two species (0.11-57.98 microg TTX/g tissue). In addition, we observed the effects of TTX or toxin extracts on the twitch contraction evoked by direct muscle stimulation of the rat hemidiaphragm or indirect phrenic nerve stimulations, in an attempt to understand the mechanisms involved in the transmission failure in the respiratory muscles, due to the ingestion of TTX bearing puffers, and found that TTX or toxin extracts preferentially affect motor nerve rather than muscle.

Inhibition of Na currents by the toxin extracts from puffer fishes captured in the sea coast of Japan.

The inhibitory effects of toxin extracted from muscle or liver of five different puffer fishes (hereafter referred as puffer(s)) captured on the Japanese sea coast were examined on voltage-dependent sodium current (I(Na)) recorded from dissociated single rat hippocampal CA1 neurons. The inhibitory effects estimated from IC(50) values of toxin extracts on I(Na) were in the order of Takifugu vermicularis > Lagocephalus wheeleri > Canthigaster rivulata > Takifugu rubripes > Arothron reticularis from muscle and T. vermicularis > T. rubripes > L. wheeleri > A. reticularis > C. rivulata from liver, thereby indicating that the amount of toxin in the liver or muscle differs between puffers. In addition, the present results indicate that the muscle of T. vermicularis, which is eaten in Japan, contains relatively higher amounts of toxin compared to those of T. rubripes, also eaten. This observation suggests that caution should be taken concerning the maximal edible amount of muscle prepared from T. rubripes.