Delirium: a symptom of how hospital care is failing older persons and a window to improve quality of hospital care.

Delirium, or acute confusional state, which often results from hospital-related complications or inadequate hospital care for older patients, can serve as a marker of the quality of hospital care. By reviewing five pathways that can lead to a greater incidence of delirium--iatrogenesis, failure to recognize delirium in its early stages, attitudes toward the care of the elderly, the rapid pace and technological focus of health care, and the reduction in skilled nursing staff--we identify how future trends and cost-containment practices may exacerbate the problem. Examining delirium also provides an opportunity to improve the quality of hospital care for older persons. Interventions to reduce delirium would need to occur at the local and national levels. Local strategies would include routine cognitive assessment and the creation of systems to enhance geriatric care, such as incentives to change practice patterns, geriatric expertise, case management, and clinical pathways. National strategies might include providing education for physicians and nurses to improve the recognition of delirium and the awareness of its clinical implications, improving quality monitoring systems for delirium, and creating environments to facilitate the provision of high-quality geriatric care.

Effects of site-directed mutations of transmembrane cysteines in sindbis virus E1 and E2 glycoproteins on palmitylation and virus replication.

The two glycoproteins that form the external spikes of the alphaviruses are type 1 membrane proteins whose transmembrane domains of hydrophobic amino acids are close to the carboxyl termini of the polypeptides and anchor the proteins in the lipid bilayer. Most of the members of the alphavirus genus contain within this transmembrane sequence one or more highly conserved cysteines, which are positioned close to the cytoplasmic face of the lipid bilayer. Cysteines in the cytoplasmic domains of the alphavirus glycoproteins and other enveloped viruses have been shown to be modified by palmitylation. To determine whether the transmembranal cysteines in Sindbis virus also were palmitylated, we used site-directed mutation to change the single transmembranal cysteine in the E1 glycoprotein and two of the transmembranal cysteines in the E2 glycoprotein to alanines. Transfection of RNA transcribed from the differently mutated Sindbis virus cDNAs led to production of infectious virus. Cells infected with the mutant virions and labeled with [3H]-palmitic acid showed that the E1 mutant no longer contained fatty acid in the E1 glycoprotein and that the extent of palmitylation was reduced about twofold in the E2 glycoprotein of virions containing the E2 mutations. At early times postinfection, the mutants grew slightly slower than the wild type in cultures of chicken embryo fibroblasts and secreted about half the amount of virus particles as wild type, but little difference was found at later time points. A triple mutant containing both the E1 and E2 mutations formed virions deficient in palmitylation of both glycoproteins, and this mutant had growth properties that were similar to those of the independent E1 and E2 mutants. Virions with the mutated glycoproteins that were deficient in fatty acid were more susceptible than the wild-type virions to inactivation by the detergent Triton X-100.

Mutations in the Sindbis virus capsid gene can partially suppress mutations in the cytoplasmic domain of the virus E2 glycoprotein spike.

Assembly and budding of alphaviruses are postulated to occur by protein-protein interactions between sites on the cytoplasmic domain of the transmembranal envelope E2 glycoprotein and on the surface of the nucleocapsid protein subunits. Genetic data to support this model have been obtained by isolating revertants of two slow-growth mutants of Sindbis virus and analyzing the sequences of the genes encoding their structural proteins. The slow-growth phenotypes of the mutants were previously shown to result from site-directed mutations of 2 amino acids in the sequence corresponding to the 33 amino acids at the carboxyl terminus of E2, which are localized to the cytoplasmic face of the plasma membrane. Putative revertants of these two mutants with faster growth rates were isolated by sequential passaging of virus grown on insect cells or chicken embryo fibroblasts. Sequence analysis of plaque-purified viruses that grew significantly better than the original mutant revealed that the original E2 mutation was present and that there were additional amino acid changes in the virus capsid. Two of the latter were introduced separately into the wild-type virus cDNA and into the genomes of the original mutants. The new strains of virus that contained both capsid and E2 mutations produced many more extracellular particles than those with the E2 mutations alone, indicating substantial suppression of the original E2 mutation. Both capsid mutations appear to be localized near a hydrophobic pocket of the capsid, which is postulated to be the site for docking of hydrophobic amino acids of the E2 cytoplasmic domain. This genetic study provides strong support for the current models of alphavirus assembly.

Metabolite changes associated with heat shocked avian fibroblast mitochondria.

A previous report from our laboratory (Collier et al 1993) showed that the elongated tubules of mitochondria in the cytoplasm of cultured chicken embryo fibroblasts collapsed to irregularly shaped structures surrounding the nuclear membrane after a 1 h heat shock treatment. The normal mitochondrial morphology reappeared upon removal of the thermal stress. We have now determined that several changes occurred in mitochondrial-related metabolites under these same heat shock and recovery conditions. Among these were significant decreases in the levels of fumarate and malate and increases in the amounts of aspartate and glutamate. In contrast, other intermediates of the tri-carboxylic acid cycle were unaltered as were levels of ATP and phosphocreatine. The changes observed might result from heat shock-induced changes in enzyme activities of the mitochondria, from alterations in the membrane-embedded specialized carrier proteins that transport metabolites between cytosol and mitochondria or from a disorganization of the electron-transport system normally coupled to oxidative metabolism. The rapid recovery, however, suggested that these changes were transient and readily reversible.

Medical professionalism under managed care: the pros and cons of utilization review.

We contend that the full consequences of managed care for American medicine and health care professionals can be more fully understood if strategies for managing care are identified-in particular, strategies for the administrative oversight of professional decision making. In this paper we apply this perspective to the study of third-party utilization review, making use of a national survey of firms contracting to provide prior authorization for hospitalization in 1992. Survey data suggest that (1) existing approaches to utilization review differ greatly among review firms; (2) review practices that might improve agency and accountability seem to be overlooked by most review firms; and (3) a large number of review firms employ practices that undermine professional autonomy in seemingly inappropriate ways.

Characterization of revertants of a Sindbis virus 6K gene mutant that affects proteolytic processing and virus assembly.

Alphaviruses of the Togaviridae encode a small hydrophobic polypeptide of 55 amino acids, noted as the 6K protein, that is synthesized as part of a polyprotein containing the sequences of the two major transmembranal viral structural glycoproteins. Mutations, insertions and deletions in the 6K appear to selectively interfere with the final stages of virus assembly and budding, producing aberrant, multi-cored infectious viruses. In addition, some of these mutations were pleiotropic and much more inhibitory to virus formation. One of the latter, a substitution of alanine in the wild-type Sindbis virus 6K gene by arginine, has been studied further and shown to interfere with normal proteolytic processing of the polyprotein. Cells infected with this mutant but not the wild-type virus also displayed viral antigens in nuclear membranes and released fragments of membranes into the cell culture media. A revertant, obtained by enrichment for a faster growing strain, 'suppressed' these defects and genetic mapping showed that the arginine codon had been modified to encode a methionine. However, the sequence of the 6K protein in this revertant was not wild-type and the revertant was still defective in assembly as demonstrated by formation of aberrant particles. A complete restoration of wild-type particle formation for this revertant could be effected by modifying the E2 glycoprotein sequence.

A pseudo-revertant of a Sindbis virus 6K protein mutant, which corrects for aberrant particle formation, contains two new mutations that map to the ectodomain of the E2 glycoprotein.

Most site-directed mutations in the gene encoding the small, membrane-associated 6K protein of Sindbis virus interfere selectively with virus assembly and budding. Particles are released that are aberrant in structure, with a single membrane enclosing multiple nucleocapsids. A revertant for the mutation that inserted a serine for a cysteine at position 39 in the 6K protein was isolated and found to correct for the defective budding so that normal particles were formed. Genetic analysis of this revertant showed that two additional mutations, which were mapped to the ectodomain of the E2 virus glycoprotein, were present in addition to the original 6K substitution. The phenotype of the revertant differed from the wild-type strain and the original mutation with regard to plaque size, thermostability, and growth in neuronal cells. Five new virus genetic constructs were prepared by insertion of these mutations into the wild-type virus. Phenotypes of these constructs confirmed that the mutations in the E2 ectodomain were responsible for both correcting the original defect in budding as well as imparting changes in cell tropism, plaque size, and thermolability on the virus. These results indicate that 6K may play an indirect role in the packing of the virus spike glycoproteins, which allows for membrane deformation and bending during the budding process.

The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process.

Alphavirus genomes encode a small hydrophobic protein of 6 kDa (the 6K protein) that is expressed as part of a large polyprotein containing the sequences of the two virus transmembranal glycoproteins which form the spikes of the infectious particle. Although made in amounts equivalent to those of the glycoproteins, very little of the 6K protein is found in secreted infectious virions. The role of this protein in virus replication and structure has been studied by use of a variety of mutationally altered forms of 6K, which yield phenotypically distinct viruses. A complete deletion of the gene encoding the 6K protein (delta 6K) of Semliki Forest Virus (SFV) has been constructed from an SFV infectious cDNA and the transcribed RNA-produced progeny virus that closely resembled the normal virus (P. Liljeström, S. Lusa, D. Huylebroeck, and H. Garoff, J. Virol. 65:4107-4113, 1991). Further studies of this mutant have now been performed, and they show that growth of delta 6K has a strong dependency on its host cell, varying from 2 to 50% of the rate of formation of the wild-type SFV. Mammalian cells are much more defective than insect and avian cells in replication of the delta 6K mutant. This mutant is not defective in formation and transport of the glycoproteins or in production of nucleocapsids, which accumulate at the plasma cell membrane in infected BHK cells. The major defect, thus, is in the final assembly and budding of new virus. In BHK cells infected with the delta 6K strain, a relatively large fraction of the total infectious virus formed can be recovered by osmotic lysis of exhaustively washed cells. Infectious SFV totally lacking 6K is identical to wild-type SFV in the early stages of virus replication, i.e., binding and uptake. The particles themselves are more thermolabile than those of wild-type SFV, suggesting that the 6K protein may be a part of the structure of wild-type virus or that the slower budding leads to an altered configuration of the trimeric spikes. These data support other studies that implicate the 6K protein as an important but nonessential component in the assembly and budding of the alphavirus particle, perhaps by affecting the packing of the glycoproteins and their interactions with membrane lipid.

How the cell copes with stress and the function of heat shock proteins.

Virtually all cells, including the prokaryotic microorganisms and the highly differentiated eukaryotic cells in human tissues, contain a small set of normally silent genes that are rapidly activated by a heat shock that raises the temperature only 5 to 10% above that of the normal physiologic range for that organism. Concomitantly, many active genes are turned off. Other kinds of stress, such as exposure to alcohol or other organic agents, heavy metals, oxidants, and agents capable of perturbing protein structure, produce a similar response, and many of these activate the same set of genes. The proteins encoded by these stress-activated genes are called heat shock proteins (hsp). They are strongly conserved in structure among widely divergent biologic species, and many function as "molecular chaperones" by forming transient complexes with partially folded or misfolded polypeptides so as to prevent their irreversible denaturation. Most hsp are members of gene/protein families, and isoforms are frequently found under normal physiologic conditions in many compartments of the cell where they act also as chaperones, binding to a variety of polypeptides to facilitate folding, oligomerization, transport, metabolic activity, and degradation. Few of the polypeptide "targets" that complex with stress-induced forms of hsp have been identified, but a number of cellular components have been shown to be particularly stress sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts.

Utilizing video-enhanced differential interference contrast microscopy of chicken embryo fibroblasts, we observed dramatic changes in the localization and morphology of mitochondria shortly after cells were subjected to a mild heat shock. At normal temperatures mitochondria were distributed in the cell cytoplasm as elongated, tubular, and dynamic organelles but upon heat shock they moved to the perinuclear region and formed a tight ring of short swollen and--in some cases--fused vesicles. Vital dye staining of mitochondria with rhodamine 123 and indirect immunofluorescence staining with antibodies against the mitochondrial-matrix protein, HSP 60, confirmed these results. Using cells double labeled with antibodies to vimentin and the HSP 60 protein, we found that the changes in mitochondria were accompanied by perturbations of the intermediate filament network that we and others have reported previously for heat shocked cells. Microtubules remained largely unaltered by our heat shock treatment and the redistribution of intermediate filaments and mitochondria occurred even in the presence of taxol, a microtubule stabilizing drug. The effects of heat shock on mitochondria were reversed when cells were returned to normal temperatures and their recovery to their normal state coincided with return of normal intermediate filament morphology. This recovery was blocked in cells treated with actinomycin D during heat shock, a result indicating that a heat shock protein may be required for recovery. These data are consistent with previously published observations that mitochondria are associated with the intermediate filament network but they extend this interaction to a cell system responding to a physiological stress normally experienced by the intact organism.

Site-directed mutations in the Sindbis virus E2 glycoprotein identify palmitoylation sites and affect virus budding.

The assembly and budding of Sindbis virus, a prototypic member of the alphavirus subgroup in the family Togaviridae, requires a specific interaction between the nucleocapsid core and the membrane-embedded glycoproteins E1 and E2. These glycoproteins are modified posttranslationally by the addition of palmitic acid, and inhibitors of acylation interfere with this budding process (M.J. Schlesinger and C. Malfer, J. Biol. Chem. 257:9887-9890, 1982). This report describes the use of site-directed mutagenesis to identify two of the acylation sites in the E2 glycoprotein as the cysteines near the carboxyl terminus of the protein which is oriented to the cytoplasmic domain of this type 1 transmembrane protein. Additional mutations were made at two prolines within a hydrophobic sequence of E2 that is highly conserved among several alphaviruses, and the mutant viruses were aberrant in assembly and particle formation. These data support earlier studies indicating that the native structure of the cytoplasmic domain of E2 is essential for proper assembly of this enveloped virus.

An in-frame insertion into the Sindbis virus 6K gene leads to defective proteolytic processing of the virus glycoproteins, a trans-dominant negative inhibition of normal virus formation, and interference in virus shut off of host-cell protein synthesis.

Encoded in the genomes of all alphaviruses is a hydrophobic polypeptide of 55 amino acids, which is post-translationally modified with 4 covalently bound palmitic acids. This protein, noted as 6K, associates with membranes and is transported along with the two virus transmembranal glycoproteins to the site of virus assembly at the infected cell's plasma membrane. Previous studies showed that mutations in the 6K protein led to the slow release of aberrant, multi-cored infectious virions. In this paper, we report that an in-frame insertion of 45 nucleotides into an internal site of the 6K gene of Sindbis virus produced single-cored infectious particles at about 5% the yield of wild-type virus when the mutant was grown on avian, mammalian, and insect cells. Although the 15 amino acids were inserted at position 29 of the 55-amino-acid 6K protein, the mutation interfered with the cotranslational proteolytic processing that cleaves the 6K at its amino terminus from the Sindbis virus p62 glycoprotein and at its carboxyl terminus from the E1 glycoprotein. As a result, the amounts of normal p62 and E1 proteins were only half that made in cells infected with wild-type virus. In addition, the post-translational proteolytic conversion of p62 to E2 occurred at 10% the rate of wild-type proteins and the extensive fatty acylation normally detected on wild-type 6K protein was not found on the altered 6K protein. None of the mutated 6K protein was detected in virions, which were morphologically indistinguishable from wild-type virus. The mutant 6K virions also were similar to wild type in their rate of attachment, uncoating, and formation of an early nonstructural virus protein in avian cells. When compared with the wild-type virus, 6K29-infected cells exhibited a decreased rate of host-cell protein synthesis shut off. However, the rates of virus capsid synthesis were the same, indicating that capsid protein, per se, is not involved in shut off of host-cell protein synthesis. In complementation studies, this mutant exhibited a trans-dominant phenotype. These data provide clues about the topology of 6K protein in the membrane and its function in virus maturation.

Inhibitory effects of HSP70 chaperones on nascent polypeptides.

Several of the major heat shock proteins (HSPs) function normally as molecular chaperones to prevent aggregation of immature polypeptides and thereby facilitate folding and oligomerization. To determine their effect on nascent polypeptides, we added purified preparations of different isoforms of HSP70 to in vitro translation reactions primed by the 26S mRNA of Sindbis virus, which encodes an autoprotease that functions cotranslationally, or by the mRNA encoding the yeast vacuolar H+ATPase, which is formed by a novel transpeptidase activity that removes the central region of the initial polypeptide. In the presence of HSP70s both the autoprotease and transpeptidase activities were inhibited, indicating that these chaperones can interact with nascent polypeptides and, in the cases studied here, perturb their normal structures.

Crystallization of Sindbis virus and its nucleocapsid.

Crystals of Sindbis virus, which contains a lipid-bilayer membrane, have been grown using polyethylene glycol. The space group is R32, a = b = 640 A, c = 1520 A. The crystals are highly mosaic, and recorded diffraction is therefore restricted to spacings of about 30 A. The crystals show that the packing of glycoproteins E1 and E2 in the icosahedral outer shell is sufficiently precise that it permits regular and repeated interactions between virus particles in the lattice. Crystals of Sindbis nucleocapsids have also been grown. The limited diffraction data are consistent with close packing of nucleocapsids 404 A in diameter.

Inhibition of influenza virus formation by a peptide that corresponds to sequences in the cytoplasmic domain of the hemagglutinin.

A decapeptide with a sequence corresponding to the cytoplasmic domain of the influenza virus hemagglutinin inhibited the release of virus particles and infectious virions when added to infected cultured cells for a 2-hr period during a one-cycle growth. Inhibition was dose-dependent in the range of 50 to 250 micrograms/ml. The peptide did not affect formation of intracellular virus-specific proteins or assembly of nucleocapsids and did not inhibit replication of two unrelated enveloped RNA viruses, Sindbis virus and vesicular stomatitis virus. Peptides of similar size but different in sequence were ineffective. We postulate that this peptide acts as a competitive inhibitor for virus-specific protein-protein interactions between the hemagglutinin and the matrix protein or nucleocapsid during virus assembly. These data offer an approach to the development of antiviral drugs based on virus specific activities.

Site-directed mutations in Sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding.

Site-directed mutagenesis was used to obtain four mutants with amino acid replacements in the cytoplasmic domain of the E2 glycoprotein and three with replacements in the 6K protein of Sindbis virus. All but one of these mutants yielded progeny virus after transfection of chicken embryo fibroblasts with RNA prepared by in vitro transcription of the virus cDNA; however, even this nonproducer mutant made virus structural proteins in the transfected cells. The other six mutants divided into two groups based on growth in chicken embryo fibroblasts. One group of four mutants (two in E2 and two in 6K) was indistinguishable from wild-type in formation of infectious virus in avian cells while the other group, consisting of two mutants, grew significantly slower. All six mutants grew slower than the parental wild-type virus in mosquito cells. In avian cells, all mutants produced extracellular particles at a slower rate than the wild-type and many of the particles contained multiple nucleocapsids, based on electron microscopy and kinetics of thermal inactivation. One of the E2 mutants with a cysteine changed to alanine and the 6K mutant with four cysteines replaced were deficient in covalent-bound palmitic acid. Two mutants with changes near the signalase cleavage sites between E2 and 6K and between 6K and E1 appeared to be defective in proteolytic processing. Despite individual differences, all of these mutants and the two previously described produced similar phenotypes in which multicored infectious virus particles were released more slowly from mosquito cells than from avian cells.

Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae.

A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae. Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure. Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein. The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography. The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents. The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis.