Efficient in vitro folding of the three-disulfide derivatives of hen lysozyme in the presence of glycerol.

Four derivatives of hen lysozyme, each lacking one native disulfide bond of the four in authentic lysozyme, were produced in Escherichia coli by expressing synthetic mutant genes. In the reoxidation reaction of the reduced derivatives purified from inclusion bodies, the addition of glycerol significantly enhanced the efficiency of folding and 'correct' disulfide bond formation. This enabled simple chromatographical purification of refolded materials. Purified 3SS-derivatives all showed lytic activities and secondary structures comparable to authentic lysozyme, which directly showed that none of the four native disulfide bonds is a prerequisite for 'correct' in vitro folding.

Characteristics of tooth pulp-driven neurons in the posterior group of the cat thalamus.

This investigation was designed to determine the responses of neurons in the posterior group of nuclei (PO) to tooth pulp stimulation. Eighteen tooth pulp-driven (TPD) neurons were recorded in 9 cats anesthetized with nitrous oxide and halothane, 14 of them in the medial part (POM) and the remainder in the lateral part (POL) of the posterior nuclei. These TPD neurons also responded to non-noxious tactile stimuli of the orofacial region of the body. Most TPD neurons responded with a short latency of less than 20 ms to tooth pulp stimulation (mean 13.5 +/- 5.9). The number of teeth having afferents to these neurons was 4-8 (mean 6.7 +/- 1.3).

[Presynaptic augmentation induced by NaF in sympathetic ganglion of bullfrog].

Effects of NaF on the synaptic transmission of bullfrog sympathetic ganglia were studied by extra- and intracellular recordings. The results obtained were as follows: 1) The amplitude of the orthodromic compound action potential (CAP) evoked by preganglionic nerve stimulation was remarkably augmented with 10 microM NaF, whereas that of the antidromic CAPs remained unchanged with the same dose of NaF. The low amplitude of the orthodromic CAP which was diminished by a low-Ca2(+)-ringer's solution reversed with an additional administration of NaF. The amplitudes of the orthodromic CAPs were enhanced by phosphodiesterase inhibitors such as isobutylmethylxanthine, theophylline, and physostigmine. In addition, augmentation of the orthodromic CAPs was induced by an adenylate cyclase activator (forskolin) and d.b-cAMP; however, its augmented responses were not affected by an additional administration of NaF. 2) In the intracellular recording, NaF showed no effect on the resting membrane potential and depolarizing response induced by acetylcholine. However, the EPSP appearing in the phase of afterhyperpolarization of the orthodromic action potential was significantly increased by NaF, whereas no effect was found on the antidromic action potential. In order to evaluate these findings, effects of NaF on the decreased low-Ca2(+)-action potential were observed. After application of NaF, the low-Ca2(+)-orthodromic EPSPs were reversed, and when the height of the EPSP was raised to the critical firing level, a spike potential was driven in the cell. These facts suggest that the site of NaF action seems to exist in the presynaptic rather than postsynaptic process. Furthermore, it suggests that NaF probably acts on Gs-protein which activates adenylate cyclase at the presynaptic membrane. This resulted in a great increase in intracellular cAMP at the synaptic terminal and it triggered the Ca2(+)-increase. As an inevitable consequence, release of transmitter from the nerve terminals of the frog sympathetic ganglion was finally facilitated. These factors supposedly resulted in augmentation of the amplitude of the orthodromic CAP.

Relationship between the optimal temperature for oxidative refolding and the thermal stability of refolded state of hen lysozyme three-disulfide derivatives.

The temperature dependence of the efficiency of oxidative refolding was examined for hen lysozyme three-disulfide derivatives produced in Escherichia coli. Each derivative was designed to lack one of the four disulfide bridges in authentic lysozyme: delta 1 (Cys6-->Ser, Cys127-->Ser), delta 2 (Cys30-->Ser, Cys115-->Ser), delta 3 (Cys64-->Ser, Cys80-->Ser), delta 4 (Cys76-->Ser, Cys94-->Ser), delta 2Ala (Cys30-->Ala, Cys115-->Ala), and delta 4Ala (Cys76-->Ala, Cys94-->Ala). The optimal refolding temperature was lowest for delta 1 (19 degrees C) and highest for delta 4Ala (30 degrees C). The chromatographically purified, completely refolded three-disulfide species were not stable above the optimal refolding temperature in the presence of glutathione. The stability of each of them was determined from the far-UV CD thermal denaturation measurement at pH 3.9 in the absence of glutathione, where the denaturation was reversible. The transition temperature was lowest for delta 1 and highest for delta 4Ala. Precise values of difference in the transition temperature among the three-disulfide derivatives were found to correlate with those in the optimal refolding temperature. Next, the effect of glycerol, which has been shown to increase the refolding efficiency [Sawano et al. (1992) FEBS Lett. 303, 11-14], was examined for delta 1 in detail. The optimal temperature for refolding increased by 3-4 degrees C with the increase in glycerol concentration by 10%. The amount of increase in the optimal refolding temperature was nearly equal to the amount of the increase in thermal stability in the presence of glycerol of refolded and purified delta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular design of lipophilic disalicylic acid compounds with varying spacers for selective lead(II) extraction.

Lipophilic disalicylic acids 5,5'-decyl-2,2'-[1,2-ethanediylbis(oxy)]bisbenzoic acid (1), 5,5'-decyl-2,2'-[1,3-propanediylbis(oxy)]bisbenzoic acid (2), 5,5'-decyl-2,2'-[oxybis(1,2-ethanediyl-oxy)]bisbenzoic acid (3), 3,5-bis[2'-(2''-carboxyphenoxy)ethyl]-4-oxahexacyclo-[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane (4), and 1,3-bis[2'-(2''-carboxyphenoxy)ethyl]adamantane (5) are evaluated as selective Pb(II) extractants. The solvent extraction of Pb(II) and of Cu(II) from buffered aqueous solutions of varying pH into chloroform by ligands 1-5 is examined in relation to the molecular structure of the dicarboxylic acid extractant. Ligand 1, with an ethylene spacer between two lipophilic salicylic acid units, exhibits excellent extraction selectivity for Pb(II) over Cu(II). Lengthening the spacer in ligands 2 and 3 diminishes both the extraction efficiency and selectivity. Ligands 4 and 5, with rigid spacer units, show significant reductions in both Pb(II) and Cu(II) extraction. Slope analysis reveals that ligand 1 reacts in a 2:1 stoichiometry with Pb(II) in extraction, which differs from the 1:1 stoichiometries for 2 and 3. The differences in the half extraction pH (DeltapH(1/2)) values for Pb(II) and Cu(II) extraction are 1.29, 0.49, and 0.48 for 1-3, respectively.