Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology最新文献

1. Both the Snell dwarf mouse (dw) and the growth hormone-deficient Little mouse (lit) exhibit a microcephalic cerebrum with hypomyelination, retarded neuronal growth, and underdevelopment of axons and dendrites. 2. The hypomyelination is due to arrested glial proliferation, suggesting that the action of growth hormone on the proliferation and maturation of both glial and neuronal cells is a necessary precondition of myelin formation, apart from the complementary or synergistic actions of thyroxine. 3. In contrast, the cerebral hypomyelination present in the inherited hypothyroid mouse (hyt) is not related to arrested glial proliferation, demonstrating that thyroid hormones can act independently on myelinogenesis.

A neurotoxin (JSTX) was isolated from the venom of spider (Nephila clavata). JSTX blocked both the excitatory postsynaptic (EPSPs) and glutamate-induced potentials in lobster neuromuscular synapse and squid giant synapse. In mammalian central nervous system, JSTX blocked the EPSPs in CA1 pyramidal neurons resulting from stimulation of Schaffer collateral/commissure input. Pharmacological investigation showed that JSTX preferentially suppressed quisqualate/kainate receptor subtypes but was much less effective on NMDA receptor. Using synthesized spider toxins we studied the structure-activity relationship and found that the 2,4 dihydroxyphenylacetyl asparagine in the toxin structure was responsible for suppressive action, while the remaining part containing a polyamine was related to the agonist binding site with the polycationic part enhancing the toxic activity. Labeling of synthesized JSTX was used for histochemical as well as biochemical studies. Using autoradiography, 125I-JSTX-3 was found to bind at the lobster neuromuscular synapse. Histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat cerebellum and hippocampus. JSTX-3-binding protein was purified from rat brain by affinity chromatography. SDS-PAGE of the affinity purified protein showed at least 4 bands ranging from 40 to 70 kDa.

1. Effect of bunazosin, an alpha 1-adrenoceptor antagonist, upon the slow inward Ca2+ current (ICa) was studied in single ventricular myocytes of the guinea-pig using a whole-cell patch-clamp technique. 2. Bunazosin (10-100 microM) decreased ICa in a concentration-dependent manner with an IC50 of 60 microM during depolarization to + 10 mV from the holding potential of -40 mV. 3. As for the inactivation process of ICa, the steady-state inactivation (f infinity) curve was shifted toward more negative potentials from -12 mV to -17 mV and -21 mV at f infinity = 0.5, by 30 microM and 70 microM bunazosin. 4. Inhibition of ICa by the compound (10 microM) was also dependent on stimulation frequency, with greater block induced at 2 Hz (50%) than at 0.33 Hz (13%). 5. It is concluded that bunazosin possesses a direct Ca2+ channel-blocking (class 4) action in a rate-dependent fashion.

1. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. 2. The structures of TH from various species have been elucidated. 3. We have cloned and determined the sequences of four types of human TH cDNA and human TH genomic DNA. 4. We have compared the amino acid sequences of TH from various species. 5. The results indicate that the amino acid sequences of TH are highly conserved among various species, and that TH consists of the regulatory domain containing serine residues which are phosphorylated by protein kinases and of the catalytic domain where the substrates, tyrosine and oxygen, and the cofactor, tetrahydrobiopterin, are bound. 6. Comparison of amino acid sequences among TH from various species can give us useful information on the functional importance of each amino acid residue.

1. Five species of Mytilus inhibitory peptides, MIP1-5, were isolated from acetone extracts of the anterior byssus retractor muscle (ABRM) of Mytilus edulis. MIP1 and MIP2 were shown to be S2-MIP and A2-MIP, respectively, first isolated from the pedal ganglia of the animal. 2. All the five peptides had a common C-terminal structure of -Pro-Xaa-Phe-Val-NH2, which was shown to be important for their biological activity. 3. The five MIPs showed similar inhibitory effects on contractions of the ABRM but did not affect catch tension and its relaxation. 4. In addition to the MIPs, catch-relaxing peptide (CARP) was also found in the ABRM.

1. During pentylenetetrazol-induced bursting activity which is characteristic intracellular potential change of seizure discharge, intracellular stored calcium is released and moved toward the inner surface of the cell membrane. Calcium is released from lysosome-like granules with morphological changes. During bursting activity, the intracellular free calcium level was higher than the normal state. During bursting activity, intracellular protein of 5 kDa and 15 kDa was changed qualitatively and quantitatively. 2. Primary cultured cerebral cortical neurons of rats and mice showed spontaneous regular firing, and by PTZ application, showed bursting activity. A single potassium channel showed the random open-close state in the normal state and showed burst type open-close state after PTZ application. 3. Primary cultured cerebral cortical neurons of the E1 mouse, the epilepsy animal model, showed developmental defects in neurite extension and content of gangliosides, in addition to their very high susceptibility to convulsions. 4. A new antiepileptic drug, TJ-960, which originates from a mixture of nine herbal drugs, normalized the above-mentioned seizure-related, calcium-related intracellular pathological phenomena. TJ-960 normalized cytochalasin-B-induced looping phenomena and protected the neuron damage induced by cytochalasin B in addition to anticonvulsant action. TJ-960 also completely normalized the cobalt-induced EEG changes and also protected against neuron damage in the hippocampus induced by cobalt application to the cerebral cortex. TJ-960 normalized the developmental defects of the E1 mouse neuron. 5. For better therapy of epilepsy, it is probably necessary to normalize the developmental defects and to protect against neuron damage in addition to inhibition of seizure activity.

1. Previous studies from our laboratory demonstrated that subcultured astroglia enhance neurite outgrowth and survival of cultured neurons from embryonic rat cerebral cortex, but suppress proliferation of neuroblasts. 2. In the present study, the mechanisms of these three effects were further investigated. 3. Dissociated neurons were seeded on poly-L-lysine-coated coverslips which were plated on subcultured astroglia, and the survival, proliferation and neurite outgrowth of the neurons were investigated. Under these conditions, survival and antimitotic effects were also observed, while neurite extension was not stimulated. 4. The results clearly indicate that neuronal survival and proliferation are regulated by soluble factors produced by astroglia. 5. We also postulated that the neurite-promoting effect of astroglia is mediated by cell-cell contact. 6. This idea was confirmed by the finding that neurite extension was enhanced when the neurons were cultured directly on heat-treated astroglia. 7. The neurite-promoting effect was found to be specific to astroglia. 8. We preliminarily characterized the astroglial surface neurite-promoting factors (ASNPFs). 9. The relationship of laminin to ASNPFs was examined by using antibody to laminin. Laminin antibody did not inhibit the ASNPF activity. 10. The effect of digestion of heat-treated astroglia with enzymes (sialidase and endo-beta-galactosidase) on the ASNPF activity was also examined. 11. These enzyme treatments did not inhibit the ASNPF activity. 12. These results suggest that enhancement of the neurite-promoting activity is not associated with the sugar moiety of ASNPFs.

1. We have isolated a neuroexcitatory tetrapeptide having a D-phenylalanine (Gly-D-Phe-L-Ala-L-Asp) from the ganglia of Achatina fulica Férussac. This peptide was termed achatin-I (Kamatani et al., 1989). In the present report, we shall present highlights from the original paper concerning the process of peptide isolation and the examination of its effects. 2. From the ganglia of about 30,000 animals, we obtained 50 micrograms of achatin-I and 17 micrograms of its stereoisomer consisting of only L-amino acid residues (Gly-L-Phe-L-Ala-L-Asp) which was termed achatin-II. The data of instrumental analyses (1H-NMR, SIMS, CD and HPLC) of isolated achatin-I and achatin-II were identical to those of synthetic ones. 3. Achatin-I showed marked excitatory effects on the three Achatina giant neurones, PON (periodically oscillating neurone), TAN (tonically autoactive neurone) and v-RCDN (ventral-right cerebral distinct neurone), whereas achatin-II had no effect. Among their stereoisomers, [D-Ala3]-achatin-I (Gly-D-Phe-D-Ala-L-Asp) had slight excitatory effects on the Achatina neurones tested. Amide derivatives of achatin-I and achatin-II were ineffective. 4. Dose-response curves of achatin-I and [D-Ala3]-achatin-I for producing the inward current of PON were measured under voltage clamp at a holding membrane voltage (Vh) of -50 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The involvement of protein phosphorylation in the pentylenetetrazole (PTZ)-induced bursting activity (BA) was evaluated in identified neurons of the snail. Euhadra peliomphala by examining the effect of various protein kinases and their inhibitors on the membrane properties induced by PTZ. 2. In neurons which normally exhibited spontaneous regular firing, PTZ elicited BA, the negative slope resistance (NSR) in the steady-state current (I)-voltage (V) relationship and a reduction of the delayed potassium current (IKD) in a dose-dependent manner. These were inhibited by the cAMP-dependent protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide. 3. Intracellular injection of catalytic subunit (CS) of cAMP-dependent protein kinase enhanced PTZ-induced NSR and reduction of IKD, as well as a conversion of the BA to a long-lasting depolarization of the membrane, whereas a saturating dose of the CS occluded PTZ action on the NSR and IKD. 4. Ca2+/calmodulin-dependent protein kinase II (CaMKII), when intracellularly injected during the depolarizing phase of PTZ-induced bursting cycle, changed to a prolonged hyperpolarization of the membrane. This kinase also restored the PTZ-suppressed IKD nearly to the pre-PTZ level. However, when intracellular injection of CaMKII and application of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin inhibitor, to the inside and outside of the neuron were simultaneously carried out, neither post-burst hyperpolarization nor restoration of the IKD was observed. 5. Intracellular injection of calmodulin, together with calcium chloride, had little effect on both the BA and reduction of IKD induced by PTZ. 6. Simultaneous application of 40 microM 1-(5-isoquinolinsulfonyl)-2-methylpiperazine, which selectively suppressed the phosphatidylserine-dependent protein phosphorylation in extracts from Euhadra ganglia, to both the inside and outside of the neuron, did not produce any significant change in the membrane properties induced by PTX. Intracellular injection of protein kinase C also brought about no effect. 7. These findings suggest that PTZ stimulates cAMP-dependent protein phosphorylation which, in turn, is involved in the development of NSR and reduction of IKD, leading to the depolarization of the membrane. In addition, we propose that the Ca2+ ions, increased during the depolarizing phase of the BA cycle, form a Ca2+/calmodulin complex and subsequent protein phosphorylation, coupled with the opening of potassium channels, leading to the membrane hyperpolarization.

1. Multiple distinct affinity states or sites of substance P (SP) receptors exist in freshly-prepared rat brain membranes. 2. Substance P receptors may couple with islet-activating protein (pertussis toxin) sensitive GTP-binding protein(s). 3. Substance P receptors may be regulated Mg2+ and Na+ in an opposite manner. 4. Some important factor(s), in addition to GTP-binding protein, appear to be involved in SP binding activity. 5. An apparent molecular weight of the SP binding site is approximately 46,000 Da.