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The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.

The search for the mechanism of action of improgan (a non-opioid analgesic) led to the recent discovery of CC12, a compound which blocks improgan antinociception. Since CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (icv) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) potently inhibited improgan antinociception in rats at doses which were inactive alone. MW06-25, a new P450 inhibitor which combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H 3 receptors, MW06-25 showed no activity at these sites, yet retained potent P450-inhibiting properties. The P450 hypothesis was also tested in Cpr low mice, a viable knock-in model with dramatically reduced brain P450 activity. Improgan (145 nmol, icv) antinociception was reduced by 37-59% in Cpr low mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, icv) abolished improgan action (70-91%) in control mice, but had no significant effect in Cpr low mice. Thus, improgan's activation of bulbospinal non-opioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which 1) improgan activates an unknown receptor to trigger downstream P450 activity, and 2) brainstem epoxygenase activity is a point of convergence for opioid and non-opioid analgesic signaling.

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This study utilized four plants of a large manufacturing organization to assess what impact implementing a Quality Circle program has on employee task and affective outcome response perceptions. The literature (nonempirical) supports the theory that employees' attitudes would improve following QC implementation. The Job Diagnostic Survey was used to obtain before/after measures on two plants that received Quality Circle intervention. In addition, two plants that did not receive QCs were used as comparison groups. The results indicated that there were no significant changes in employee perceptions following QC implementation. However, a trend analysis found a marginal decline in the variables, in that eight of twelve declined after QC implementation.