New technique for measuring dynamic axonal transport and its application to temperature effects

A new technique was devised for the dynamic detection of the axoplasmic transport of β-radioactively labeled materials in which a semiconductor radiation detector was used as the β-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the β-radioactive distribution of axoplasmic transport could be measured in an axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 °C. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 °C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q₁₀ is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. This technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

Studies on antiviral glycosides. II. Mode of action for virucidal effects on Sendai virus

Treatment of Sendai virus with $\text{p-(sec-}\text{butyl)-phenyl-6-chloro-6-deoxy-β-}\text{d}\text{-glucopyranoside}$, followed by freezing and thawing resulted in a loss of hemolytic and cell fusion activities as well as infectivity without affecting hemagglutinating and neuraminidase activities. The anti-hemolytic activity of this compound was reversed by the addition of phosphatidyl choline to the virus samples. $\text{p-}\text{Azidophenyl-6-chloro-6-deoxy-β-}\text{d}\text{-[}{}^3\text{H]glucopyranoside}$ was successfully used for photoaffinity labeling of a specific virion site, and we confirmed the affected site of the glucoside to be the lipid components in the viral envelopes.     

Microtubules inside the plasma membrane of squid giant axons and their possible physiological function

Summary The effects of application of the microtubule-disassembling reagents to squid giant axons upon resting potential, the height of the propagated action potential, and the threshold to evoke action potential were studied using colchicine, podophyllotoxin, vinblastine, griseofulvin, sulfhydryl reagents including NEM, diamide, DTNB and PCMB, and Ca²⁺ ions. At the same time, the effects of concentrations of K halides and K glutamate on the above physiological properties were studied in comparison within vitro characteristics of microtubule assembly from purified axoplasmic tubulin.It was found that there was good correlation between conditions supporting maintenance of membrane excitability and microtubule assembly. The experiments suggest that associated with the internal surface of the plasma membrane there are microtubules which regulate in part both resting and action potentials.     

New technique for measuring dynamic axonal transport and its application to temperature effects.

A new technique was devised for the dynamic detection of the axoplasmic transport of beta-radioactively labeled materials in which a semiconductor radiation detector was used as the beta-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the beta-radioactive distribution of axoplasmic transport could be measured in a axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 degrees D. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 degrees C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q10 is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. this technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues

The substrate specificity of rat liver microsomal glutathione transferase toward glutathione has been examined in a systematic manner. Out of a glycyl-modified and eight gamma-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, alpha-L-Glu-L-Cys-Gly, alpha-D-Glu-L-Cys-Gly, and gamma-L-Glu-L-Cys-beta-Ala) function as substrates. The kinetic parameters for three of these substrates (the alpha-D-Glu-L-Cys-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the N-ethylmaleimide-activated microsomal glutathione transferase. The alpha-L-Glu-L-Cys-Gly analogue is similar to GSH in that it has a higher kcat (6.9 versus 0.6 s-1) value with the activated enzyme compared with the unactivated enzyme but displays a high Km (6 versus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kcat (8.9 versus 6.7 s-1) with the N-ethylmaleimide-treated enzyme but retains a higher Km value (50 versus 15 mM). Thus, the alpha-amino group of the glutamyl residue in GSH is important for the activity of the activated microsomal glutathione transferase. These observations were quantitated by analyzing the changes in the Gibbs free energy of binding calculated from the changes in kcat/Km values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the alpha-amino group of the glutamyl residue in GSH contributes 9.7 kJ/mol to catalysis by the activated enzyme, whereas the corresponding value for the unactivated enzyme is 3.2 kJ/mol. The importance of the acidic functions in glutathione is also evident as shown by the lack of activity with 4-aminobutyric acid-L-Cys-Gly and the low kcat/Km values with gamma-L-Glu-L-Cys-beta-Ala (0.03 and 0.01 mM-1s-1 for unactivated and activated enzyme, respectively). Utilization of binding energy from a correctly positioned carboxyl group in the glycine residue (10 and 17 kJ/mol for unactivated and activated enzyme, respectively) therefore also appears to be required for optimal activity and activation. A conformational change in the microsomal glutathione transferase upon treatment with N-ethylmaleimide or trypsin, which allows utilization of binding energy from the alpha-amino group of GSH as well as the glycine carboxyl in catalysis, is suggested to account for at least part of the activation of the enzyme.     

Anticalmodulin drugs block the sodium gating current of squid giant axons

Summary The effects of calmodulin (CaM) antagonists (W-7, W-5, trifluoperazine, chlorpromazine, quinacrine, diazepam, propericyazine and carmidazolium) on the sodium and potassium channels were studied on the intracellularly perfused and voltage-clamped giant axon of the squid. It was found that the drugs are more potent blockers of the sodium current than of the potassium current. The drugs also reduce the sodium gating current. The blockage of the sodium and gating current can be explained by assuming that the drugs interact with the sodium gating subunit in one of its closed states. The site of action is probably the intracellular surface of the axolemma where presumably a Ca²⁺-calmodulin complex can be formed.     

Water insoluble fraction of egg yolk maintains porcine sperm motility by activating adenylate cyclase.

The decrease in motility of porcine cauda epididymal sperm was less than that of caput epididymal sperm in the medium containing bicarbonate. This may be due to the difference of sensitivity of adenylate cyclase to bicarbonate between mature and immature sperm; activation of mature sperm enzyme by bicarbonate was higher than that of immature sperm. Nondialysable fraction of egg yolk prevented the decrease in motility of immature sperm in the presence of bicarbonate, but it was not effective for the motility of mature sperm under the same condition, because only bicarbonate is sufficient for the maintenance of its motility. In the absence of bicarbonate, both mature and immature sperm required egg yolk to maintain motility. The favorable effect of egg yolk on the motility is ascribed to the enhancement of intracellular cAMP level. Partial fractionation of egg yolk showed that water-insoluble lipoprotein fraction contains factor(s) which activates adenylate cyclase in sperm plasma membrane. This is the first report in which high molecular weight activator of the sperm enzyme was demonstrated.     

Effect of phosphoramidon on big endothelin-2 conversion into endothelin-2 in human renal adenocarcinoma (ACHN) cells. Analysis of endothelin-2 biosynthetic pathway.

The biosynthetic pathway of endothelin (ET)-2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET-2-related peptides, ET-2, big ET-2(1-38) and big ET-2(22-38). Phosphoramidon decreased the amount of ET-2 and increased that of big ET-2(1-38) dose-dependently. The amount of big ET-2(1-37) did not significantly change. These results suggest that big ET-2 is composed of 38 and not 37 amino acid residues, and that a putative ET-2-converting enzyme (ECE-2) should be classified as a phosphoramidon-sensitive neutral metalloprotease, bearing a resemblance to the putative ET-1-converting enzyme (ECE-1) in endothelial cells.