IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha.

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.

Delay in hearing loss following drug administration. A consistent feature of amikacin ototoxicity.

The time course of threshold increase in the VIII nerve compound action potential was studied in guinea pigs following amikacin administration at four different constant infusion rates. Despite the wide range of dosing durations required to achieve drug ototoxicity (2-24 days), the full development of both high and low frequency hearing loss was invariably found to be delayed with respect to the time of drug removal. The greatest degree of delayed hearing loss generally occurred within the first 7 days after drug removal, with smaller losses occurring during later time intervals. The delay showed a tendency to decrease as the ototoxic dose was increased. Using the data from the two highest dosing rates, it was estimated that a minimum of 4 days had to elapse before any hearing loss could be detected, once an ototoxic amount of drug had been administered. These data suggest that hearing loss is always substantially delayed with respect to the receipt of an ototoxic dose of amikacin, and that this must be taken into account when conducting animal experiments and when monitoring hearing in patients for the early detection of ototoxicity.

Incidence of amikacin ototoxicity: a sigmoid function of total drug exposure independent of plasma levels.

A sigmoid curve was found to closely describe the relationship between the incidence of amikacin ototoxicity (greater than or equal to 15 dB hearing loss at a given frequency) and either (1) total dose, or (2) the area under the curve (AUC) describing plasma drug concentration v time over the total period of amikacin administration (total AUC) in continuously infused guinea pigs. Total dose or total AUC estimates of the drug exposure required to produce ototoxicity in 50% of the animals (ED50s) were not significantly different over an eight-fold range of dosing rates or plasma concentrations. A theoretical explanation for this result is that ototoxicity occurs only when a critical amount of drug is accumulated at the ototoxic site by an essentially unidirectional process with a rate that is slow and linearly related to the extracellular drug concentration. The sigmoid relationships for pooled data were parallel in slope for all hearing frequencies from 2 to 32 kHz, and the ED50s showed a strong negative linear relationship to the log of the hearing frequency over this range. The magnitude of ototoxicity expressed as the number of octaves (frequency ratios of 2) for which hearing loss damage was continuous from 32 kHz downward, was correlated to both total dose (r = .605) and total AUC (r = 0.703). No relationship between ototoxicity and plasma level or dosing rate was found. The extreme steepness of the dose-effect curve for the incidence of ototoxicity greatly amplified the variability between individuals and offers an explanation for the unpredictability of aminoglycoside ototoxicity in human patients. The results indicate that either total dose or total AUC (in cases of highly unpredictable blood levels), and not peak or trough serum levels, should be used as an index of ototoxic risk and that the safety limits of drug exposure should be set conservatively.

A cost and production analysis of hospital dental care programs.

To provide hospital dental programs with useful information about the expansion of dental services and the identification of pertinent financial information, a production function and cost function analysis was performed. Results showed that hospital ownership (public or private) and size of the dental clinics were associated with the cost of providing dental services and the volume of services provided. Among 23 hospitals studied, private hospitals had a much lower cost per visit, had more paid attending dentist staff, paid their resident dentists less, and had significantly more billings paid by Medicaid and by patients than public hospitals. When stratified by ownership and size, these basic differences were accentuated for the small clinics. Except for primarily the Medicaid and self-pay billings, the characteristics of large public and private hospital dental clinics were extremely similar. Multiple regression analysis found that a decrease in cost per visit was associated with more visits to dentists and more to hygienists. Production of dental services could be increased by increasing the number of attending dentists, hygienists, and residents. Preliminary econometric analysis reveals that the optimal mix of attending dentists to resident dentists should be approximately 1.8 full-time equivalent (FTE) resident for every 1 attending FTE dentist to produce the most dental services at the lowest cost.

2,3-dihydro and carbocyclic analogues of tryptamines: interaction with serotonin receptors.

Several dihydro and carbocyclic analogues of tryptamine were evaluated in order to determine the role of the heterocyclic portion of the indole nucleus on the interaction of indolealkylamines with the serotonin receptors of the rat fundus. Reduction of the C2--C3 double bond or replacement of the indole nitrogen with an sp3-hybridized carbon atom results in a 50% decrease in receptor affinity. Complete removal of the five-membered ring of N,N-dimethyltryptamine reduces affinity by an order of magnitude. It appears that an intact indole nucleus, though not entirely necessary, results in an optimal receptor interaction for the indolealkylamines examined.

Synthesis and evaluation of novel alkylpiperazines as potential dopamine antagonists.

Several alkylpiperazines, monocyclic subfragments of known tricyclic neuroleptic agents, were evaluated as dopamine antagonists in the isolated rabbit ear artery preparation. Compound prepared and evaluated are of the general structure Ar-X-(CH2)n-Y, where X = C, O, and N, n = 1-3, and Y, for the most part, was 4-methylpiperazine. Those compounds where X - NH, n = 3, and X = (Z)-CH - CH, n = 2, with an electron-withdrawing group meta to the side chain, possess dopamine antagonist activity comparable to that of clozapine. It is concluded that the entire tricyclic structure of phenothiazine-like agents (or at least more than a monocyclic ring system) is necessary for optimal activity as a dopamine antagonist in the receptor preparation used in this study.

Responses of denervated adult newt limb stumps to reinnervation and reinjury.

It is well known that denervated adults newt limbs do not regenerate. Not understood is why denervated newt limb stumps fail to initiate regeneration upon reinnervation. In an effort to define the regeneration limiting factors, we examined the histology of long-term denervated newt limb stumps and tested the effects of various kinds of reinjury. In 5-week denervated limb stumps there were present dedifferentiated cells, differentiated cartilage, and densely packed layered cells. The epidermis covering the limb tip resembled skin epidermis rather than wound epidermis. The kinds of reinjury that were successful in restoring the regeneration capabilities to 5-week denervated limb stumps included; reamputation (100%), a single razor incision (60%), and removal of the healed tissues from the distal limb tip (70%). The results of the study emphasize the importance of both injury and wound epidermis in initiating regeneration.