Behavior of delayed current under voltage clamp in the supramedullary neurons of puffer

Depolarizations applied to voltage-clamped cells bathed in the normal solution disclose an initial inward current followed by a delayed outward current. The maximum slope conductance for the peak initial current is about 30 times the leak conductance, but the maximum slope conductance for the delayed current is only about 10 times the leak conductance. During depolarizations for as long as 30 sec, the outward current does not maintain a steady level, but declines first exponentially with a time constant of about 6 msec; it then tends to increase for the next few seconds; finally, it declines slowly with a half-time of about 5 sec. Concomitant with the changes of the outward current, the membrane conductance changes, although virtually no change in electromotive force occurs. Thus, the changes in the membrane conductance represent two phases of K inactivation, one rapidly developing, the other slowly occurring, and a phase of K reactivation, which is interposed between the two inactivations. In isosmotic KCl solution after a conditioning hyperpolarization there occurs an increase in K permeability upon depolarization. When the depolarizations are maintained, the increase of K permeability undergoes changes similar to those observed in the normal medium. The significance of the K inactivation is discussed in relation to the after-potential of the nerve cells.     

Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis

Key message

Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine.

Abstract

Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

     

Alteration of the kinetics of type I procollagen synthesis in human osteosarcoma cells by 1,25-dihydroxyvitamin D3

The kinetics of type I procollagen synthesis in a human osteosarcoma cell line, MG 63, were investigated after treatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂ D₃), a hormonal inducer of phenotypic differentiation. Pulse label and chase experiments demonstrated greatly enhanced production and more rapid reduction of intracellular procollagen molecules in the 1,25-(OH)₂ D₃–treated cells as compared to the nontreated case. After a chase for 1 h, labeled procollagen was reduced by nine-tenths in 1,25-(OH)₂ D₃–treated cells, while half of the radioactivity still remained in nontreated cells. The expression rate of type I collagen, which was examined by pulse label experiment, was elevated in association with an increase in the mRNA coding for the type I collagen α1 chain by 1,25-(OH)₂ D₃ treatment. However, the amount of intracellular procollagen present after 4 h continuous labeling was almost the same, independent of the 1,25-(OH)₂ D₃ treatment. Thus, we conclude that strage of the molecule was not affected. The results therefore suggest an increase in both the synthesis and secretion of type I collagen. The 1,25-(OH)₂ D₃ treatment was also found to induce the α subunit of prolyl 4-hydroxylase and to be associated with an elevated level of hydroxyproline in the procollagen. Moreover, gelatinase B–resistant procollagen molecules, indicative of intracellular procollagen molecules in the stable triple helical form, were detected only in the 1,25-(OH)₂ D₃–treated cells. These data suggest more efficient proline hydroxylation is involved in rapid secretion of procollagen after hormone administration. The present evidence points to posttranslational control of procollagen synthesis. J. Cell. Biochem. 65:542–549. © 1997 Wiley-Liss Inc.     

Correlation of Transmitter Release with Membrane Properties of the Presynaptic Fiber of the Squid Giant Synapse

Depolarization of the presynaptic terminal by current produced a postsynaptic potential (PSP) which increased with increasing presynaptic polarization and then reached a plateau. Iontophoretic injection of tetraethylammonium ions (TEA) into the presynaptic axon near the terminal produced a prolonged presynaptic spike. The resulting PSP is increased in size and its time course closely followed that of the presynaptic spike. The presynaptic fiber no longer exhibited rectification and strong depolarizations revealed that the PSP reached a maximum with about 110 mv depolarization. Further depolarization produced a decrease in PSP amplitude and finally transmission was blocked. However, a PSP then always appeared on withdrawal of the depolarizing current. Under the conditions of these experiments, the PSP could be considered a direct measure of transmitter release. Bathing the TEA-injected synapse with concentrations of tetrodotoxin (TTX) sufficient to block spike activity in both pre- and postsynaptic axons did not greatly modify postsynaptic electrogenesis. However, doubling TTX concentration reversibly blocked PSP. Thus the permeability changes to Na and K accompanying the spike do not appear necessary for transmitter release. Some other processes related to the level of presynaptic polarization must be involved to explain the data. The inhibition of transmitter release by strong depolarizations appears to be related to Ca action. A membrane Ca current may also be necessary for normal transmitter release.     

Crystal structure of the ADP-dependent glucokinase from Pyrococcus horikoshii at 2.0-A resolution: a large conformational change in ADP-dependent glucokinase

Although ATP is the most common phosphoryl group donor for kinases, some kinases from certain hyperthermophilic archaea such as Pyrococcus horikoshii and Thermococcus litoralis use ADP as the phosphoryl donor. Those are ADP-dependent glucokinases (ADPGK) and phosphofructokinases in their glycolytic pathway. Here, we succeeded in gene cloning the ADPGK from P. horikoshii OT3 (phGK) in Escherichia coli,and in easy preparation of the enzyme, crystallization, and the structure determination of the apo enzyme. Recently, the three-dimensional structure of the ADPGK from T. litoralis (tlGK) in a complex with ADP was reported. The overall structure of two homologous enzymes (56.7%) was basically similar: This means that they consisted of large alpha/beta-domains and small domains. However, a marked adjustment of the two domains, which is a 10-A translation and a 20 degrees rotation from the conserved GG sequence located at the center of the hinge, was observed between the apo-phGK and ADP-tlGK structures. The ADP-binding loop (430-439) was disordered in the apo form. It is suggested that a large conformational change takes place during the enzymatic reaction.     

Cloning and expression of a novel rat GABAA receptor

Two full-length cDNA clones encoding alpha- and beta-subunits of a GABAA receptor have been isolated from a rat cerebral cortex cDNA library. The mature alpha-subunit protein consists of 428 amino acids with a calculated Mr of 48,680. This protein is highly homologous (approximately 99% amino acid identity) with the bovine brain alpha 1-subunit receptor [(1988) Nature 335, 76-79]. The mature rat beta-subunit receptor is a 448 amino acid polypeptide and shares approximately 80% amino acid identity with the previously characterized bovine GABAA receptor beta-subunit [(1987) Nature 328, 221-227]. Co-expression of the cloned DNA in Xenopus oocytes produces a functional receptor and ion channel with pharmacological characteristics of a GABAA receptor. GABAA alpha- and beta-subunit mRNA is detectable in the cortex, cerebellum and hippocampus.