Identifying costs for capitation in psychiatric case management.

This article presents an example of how one hospital identified costs for capitation in psychiatric case management. An 18-month postacute case management pilot project collected data on a nurse-specific and patient-specific basis. Costs were identified using activity-based costing methodology.

Characterization of beta-amyloid peptide precursor processing by the yeast Yap3 and Mkc7 proteases.

Two proteases, denoted beta- and gamma-secretase, process the beta-amyloid peptide precursor (APP) to yield the Abeta peptides involved in Alzheimer's disease. A third protein, alpha-secretase, cleaves APP near the middle of the Abeta sequence and thus prevents Abeta formation. These enzymes have defied identification. Because of its similarity to the systems of mammalian cells the yeast secretory system has provided important clues for finding mammalian processing enzymes. When expressed in Saccharomyces cerevisiae APP is processed by enzymes that possess the specificity of the alpha-secretases of multicellular organisms. APP processing by alpha-secretases occurred in sec1 and sec7 mutants, in which transport to the cell surface or to the vacuole is blocked, but not in sec17 or sec18 mutants, in which transport from the endoplasmic reticulum to the Golgi is blocked. Neutralization of the vacuole by NH4Cl did not block alpha-secretase action. The time course of processing of a pro-alpha-factor leader-APP chimera showed that processing by Kex2 protease, a Golgi protease that removes the leader, preceded processing by alpha-secretase. Deletions of the genes encoding the GPI-linked aspartyl proteases Yap3 and Mkc7 decreased alpha-secretase activity by 56 and 29%, respectively; whereas, the double deletion decreased the activity by 86%. An altered form of APP-695, in which glutamine replaced Lys-612 at the cleavage site, is cleaved by Yap3 at 5% the rate of the wild-type APP. Mkc7 protease cleaved APP (K612Q) at about 20% the rate of wild-type APP. The simplest interpretation of these results is that Yap3 and Mkc7 proteases are alpha-secretases which act on APP in the late Golgi. They suggest that GPI-linked aspartyl proteases should be investigated as candidate secretases in mammalian tissues.

Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae.

Overexpression of protein disulfide isomerase (PDI) from a single chromosomally integrated copy in Saccharomyces cerevisiae results in ten-fold higher levels of secretion of human platelet derived growth factor B homodimer, and a four-fold increase in secretion of Schizosaccharomyces pombe acid phosphatase. This result provides evidence that inefficient protein folding limits the secretion of some heterologous proteins, and that manipulation of the endoplasmic reticulum lumenal environment can help overcome this limitation.

The expression and processing of human beta-amyloid peptide precursors in Saccharomyces cerevisiae: evidence for a novel endopeptidase in the yeast secretory system.

In mammalian cells, the transmembrane beta-amyloid peptide precursor (beta-APP) undergoes a complex series of alternative proteolytic processing steps that result in the secretion of varying proportions of its extra-cellular domain (protease nexin II) and beta-amyloid peptide. The protein is also reinternalized and degraded in the endosomal-lysosomal system. The relative efficiencies of these competing processes determine the yield of beta-amyloid peptide. Several proteases have been implicated in this complex processing pathway, although none has been identified to date. The yeast secretory system contains proteases homologous to mammalian pro-hormone convertases and is susceptible to genetic manipulation. We therefore investigated the expression and processing of the beta-amyloid peptide precursors (beta-APP-695 and beta-APP-751) in Saccharomyces cerevisiae transformed with human beta-APP cDNA's. beta-APP (695 or 751) cDNA either with its authentic signal sequence or the yeast-derived prepro-alpha-factor leader, was inserted into a glucose-regulated expression vector and transfected into a protease-deficient yeast strain. In all instances, expression of beta-APP was about 1% of total protein. Protease protection studies indicated that either the natural human signal sequence or the alpha-factor leader sequence targetted beta-APP to the endoplasmic reticulum and inserted it with the amino-terminal domain in the lumen. All of the beta-APP fused to the alpha-factor leader proceeded to the trans-Golgi, where Kex2 endopeptidase removed the leader and released the normal amino-terminus of beta-APP. About one-half of the beta-APP was also cleaved at the "alpha-secretase" site in the middle of the beta-peptide sequence, 12 residues before the membrane-spanning sequence. A fraction of the alpha-secretase-cleaved beta-APP appeared in the culture medium; however, most of it associated with the exterior of the cells. The carboxyl-terminal fragments formed by cleavage at the alpha-secretase site accumulated in the membranes. Other proteolytic processes generated membrane-associated carboxyl-terminal fragments that also resembled those found in mammalian cells. These results indicate that the secretory system of S. cerevisiae possesses proteases with specificities similar to the mammalian enzymes that process beta-APP.

Precursor binding to yeast mitochondria. A general role for the outer membrane protein Mas70p.

Binding of precursors to import receptors on the mitochondrial surface is one of the earliest steps of protein import into mitochondria. In yeast, one of these receptors is a 70-kDa outer membrane protein termed Mas70p. Pulse-chase studies with intact yeast cells had indicated that Mas70p accelerates the import of all mitochondrial precursors tested. In contrast, import experiments with isolated mitochondria suggested that Mas70p accelerated import of only a subset of precursors (Hines, V., Brandt, A., Griffiths, G., Horstmann, H., Brütsch, H., and Schatz, G. (1990) EMBO J. 9, 3191-3200). To resolve this discrepancy, we have now studied the interaction of Mas70p-deficient and wild-type yeast mitochondria with a precursor (pre-alcohol dehydrogenase III) whose import into isolated mitochondria is not accelerated by Mas70p under the usual assay conditions. Mas70p enhanced binding of pre-alcohol dehydrogenase III to the surface of mitochondria in which the electrochemical potential across the inner membrane had been dissipated by an uncoupler; the bound precursor could be efficiently chased into the mitochondria if the potential was restored. The precursor to cytochrome c1 was also bound to mitochondria in a Mas70p-dependent manner. Mas70p also enhanced the direct import of pre-alcohol dehydrogenase III into isolated mitochondria, provided the precursor was first denatured with urea. Under these conditions, the import rate in vitro was more similar to that in intact cells. Mas70p had no effect on the binding or the import of artificial precursors containing mouse dihydrofolate as the "mature" domain. We conclude that Mas70p is an import receptor for most, if not all authentic mitochondrial precursor proteins, but that its function is not always rate-limiting in import experiments with isolated mitochondria.

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.

Analysis of the kinetic mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase.

The steady-state kinetic mechanism of highly purified bovine liver mitochondrial dihydroorotate dehydrogenase has been investigated. Initial velocity analysis using S-dihydroorotate and coenzyme Q6 revealed parallel-line, double-reciprocal plots, indicative of a ping-pong mechanism. The dead-end inhibition pattern with barbituric acid and the reactions with alternate cosubstrates methyl-S-dihydroorotate and menadione also point to a ping-pong mechanism. However, product orotate was found to be competitive with dihydroorotate and uncompetitive with Q6. These findings suggest that dihydroorotate dehydrogenase may follow a nonclassical, two-site ping-pong mechanism, typical of an enzyme that contains two non-overlapping and kinetically isolated substrate binding sites. That these two sites communicate by an intramolecular electron-transfer system involving FMN and perhaps an iron-sulfur center is also suggested by the kinetic behavior of the enzyme.

Mechanistic studies on the bovine liver mitochondrial dihydroorotate dehydrogenase using kinetic deuterium isotope effects.

Dihydroorotates deuteriated at both C5 and C6 have been prepared and used to probe the mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase. Primary deuterium isotope effects on kcat are observed with both (6RS)-[5(S)-2H]- and (6RS)-[6-2H] dihydroorotates (3 and 6, respectively); these effects are maximal at low pH. At pH 6.6, DV = 3.4 for the C5-deuteriated dihydroorotate (3), and DV = 2.3 for the C6-deuteriated compound (6). The isotope effects approach unity at pH 8.8. Analysis of the pH dependence of the isotope effects on kcat reveals a shift in the rate-determining step of the enzyme mechanism as a function of pH. Dihydroorotate oxidation appears to require general base catalysis (pKB = 8.26); this step is completely rate-determining at low pH and isotopically sensitive. Reduction of the cosubstrate, coenzyme Q6, is rate-limiting at high pH and is isotopically insensitive; this step appears to require general acid catalysis (pKA = 8.42). The results of double isotope substitution studies and analysis for substrate isotope exchange with solvent point toward a concerted mechanism for oxidation of dihydroorotate. This finding serves to distinguish further the mammalian dehydrogenase from its parasitic cognate, which catalyzes a stepwise oxidation reaction [Pascal, R., & Walsh, C.T. (1984) Biochemistry 23, 2745].

Purification and properties of the bovine liver mitochondrial dihydroorotate dehydrogenase.

Dihydroorotate dehydrogenase has been purified 6,000-fold from bovine liver mitochondria to apparent homogeneity in six steps. Electrophoretic migration of the homogeneous enzyme on sodium dodecyl sulfate-polyacrylamide gels reveals a subunit Mr of 42,000. By contrast to the well-characterized, cytosolic dihydroorotate oxidases (EC 1.3.3.1), the purified bovine dehydrogenase is a dihydroorotate:ubiquinone oxidoreductase. Maximal rates of orotate formation are obtained using coenzymes Q6 or Q7 as cosubstrate electron acceptors. Concomitant with substrate oxidation, the enzyme will reduce simple quinones, such as benzoquinone, but at significantly lower rates (10-15%) than that obtained for reduction of coenzyme Q6. Enzyme-catalyzed substrate oxidation is not supported by molecular oxygen. The specificity of the purified enzyme for dihydropyrimidine substrates has also been explored. The methyl-, ethyl-, t-butyl-, and benzyl-S-dihydroorotates are substrates, but 1- and 3-methyl and 1,3-dimethyl methyl-S-dihydroorotates are not. Competitive inhibitors include product orotate, 5-methyl orotate, and racemic cis-5-methyl dihydroorotate.

Radiation safety and quality assurance in North American dental schools.

A survey of North American dental schools revealed that processing quality control and routine maintenance checks on x-ray generators are, in most instances, being carried out in a timely manner. Available methods for reducing patient exposure to ionizing radiation are, however, not being fully implemented. Furthermore, in some instances, dental students are still being exposed to x-rays primarily for teaching purposes.