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Biomedical subjects

J King

Publications and source records attributed to J King.

At least 217 records · Page 12Linked to original sources
PubMed1993

Folding of the phage P22 coat protein in vitro.

Within infected Salmonella cells, newly synthesized 47-kDa phage P22 coat polypeptides fold without covalent modifications into assembly-competent subunits. Coat protein subunits interact with scaffolding protein to form the icosahedral procapsid precursor of the mature, T = 7, virions. In these lattices, the coat subunits form seven classes of local bonding interactions [Prasad, B. V. V., Prevelige, P. E., Marieta, E., Chen, R. O., Thomas, D., King, J., & Chiu, W. (1993) J. Mol. Biol. 231, 65-74]. Coat protein denatured in guanidine hydrochloride could be refolded to soluble, monomeric subunits by rapid dilution into buffer at concentrations of protein up to 25 micrograms/mL. The fluorescence emission spectrum of soluble coat protein monomers was between that of the assembled shells and the denatured protein, suggesting the presence of tryptophans at the subunit interfaces in the shells. Kinetic studies of the refolding of coat protein revealed an intermediate whose continued folding could be inhibited by the hydrophobic dye bisANS. The kinetic intermediate bound 10.80 +/- 1.20 bisANS molecules while the folded monomer bound 1.24 +/- 0.36 bisANS molecules. When coat polypeptide chains were refolded at 50 micrograms/mL, aggregation competed with folding. Aggregation of the folding intermediates increased in the presence of bisANS. The kinetic folding intermediate that binds bisANS probably represents the species at the junction of the productive pathway to soluble and assembly-competent coat monomers and the off-pathway steps to inclusion bodies. The relationship between these soluble monomers and the conformations observed in the T = 7 lattice remains unclear.

Anilino Naphthalenesulfonates↗
PubMed1993

Temperature-sensitive mutations and second-site suppressor substitutions affect folding of the P22 tailspike protein in vitro.

One of the central problems in protein folding is how amino acid sequences within polypeptide chains direct polypeptide chain folding and avoid off-pathway aggregation both in intracellular environments and in the test tube. The tailspike protein of phage P22 is a model system for which genetic analysis has permitted mutational dissection of the role of amino acid positions in the polypeptide chain in directing its in vivo folding. Two classes of mutations that affect intracellular folding and aggregation have been characterized; temperature-sensitive folding (tsf) mutants and second-site suppressors of tsf mutants. Here we report the effects of these mutations on the in vitro refolding and aggregation pathway of the purified proteins. The tsf mutations reduced refolding yields at high temperature and increased aggregation, while second-site suppressors enhanced refolding and inhibited aggregation in the test tube. For both types of mutations, the strength of the effects observed in vitro correlated with their in vivo phenotypes. The results confirm that the mutations act intrinsically on the folding pathway of the tailspike polypeptide and not through accessory proteins.

Amino Acid Sequence↗
PubMed1993

Three-dimensional transformation of capsids associated with genome packaging in a bacterial virus.

Three-dimensional structures of the empty procapsid and the mature capsid of the Salmonella bacteriophage P22 have been determined to a resolution of 28 A using electron cryomicroscopy and computer image processing. The coat subunits in both the structures are arranged as pentamers and hexamers on a T = 7 icosahedral lattice. The two structures display significant differences in shape, size and intersubunit interactions. The empty procapsid is spherical in contrast to the distinctly larger and polyhedral mature capsid. The empty procapsid structure exhibits holes at all the quasi sixfold positions that are absent in the mature capsid. These holes may be the exit ports for scaffolding subunits. Detailed comparisons of the two structures indicate that extensive structural changes take place during maturation in all seven quasi-equivalent subunits. These changes cause flattening of the icosahedral facets, capsid expansion and closing of the holes. This process results in a stable and impenetrable capsid that protects the bacterial genome.

Bacteriophage P22↗
PubMed1993

Temperature-sensitive mutations in the phage P22 coat protein which interfere with polypeptide chain folding.

Temperature-sensitive mutations in the coat protein of phage P22 severely restrict formation of infectious particles at restrictive temperature. A set of 25 temperature-sensitive strains, which had been localized to regions of the coat gene (Casjens, S., Eppler, K., Sampson, L., Parr, R., and Wyckoff, E. (1991) Genetics 127, 637-647), define 17 sites of single amino acid substitutions by DNA sequencing. Particles assembled from the mutant proteins at permissive temperature were not thermolabile at restrictive temperature, nor defective in the infectious process. At restrictive temperature, ts mutant polypeptide chains were synthesized at near wild-type rates. These inactive chains were not degraded within the cells. The ts chains failed to interact with scaffolding proteins to form the procapsid precursor shell, and they did not polymerize with each to form aberrant shells. Rather, the mutant coat protein accumulated as insoluble aggregates, with the amorphous morphology of inclusion bodies. The results indicate that the chains fail to reach the conformation needed for subunit-subunit or subunit-scaffolding interaction. These mutations appear to be of the class of temperature-sensitive folding mutations, which destabilize an intermediate in the intracellular folding pathway.

Amino Acid Sequence↗
PubMed1993

Proteus mirabilis biofilms and the encrustation of urethral catheters.

Bacterial biofilms were observed on 69 of 75 catheters taken from patients undergoing long-term bladder management. Ten catheters were colonized by pure cultures of Proteus mirabilis. In each of these cases the bacteria formed layers on the catheter surface, underlying encrustations of struvite and hydroxyapatite which partially or completely occluded the catheter lumen. Encrustation was also apparent on catheters colonized by P. mirabilis plus other species, but was rarely seen on catheters colonized by non-urease-producing species. These observations support the hypothesis that catheter encrustation is brought about by the activity of urease-producing biofilms and confirms that the main target in the control of catheter encrustation should be P. mirabilis.

Catheterization↗
PubMed1993

Conformational transformations in the protein lattice of phage P22 procapsids.

During the packaging of double-stranded DNA by bacterial viruses, the precursor procapsid loses its internal core of scaffolding protein and undergoes a substantial expansion to form the mature virion. Here we show that upon heating, purified P22 procapsids release their scaffolding protein subunits, and the coat protein lattice expands in the absence of any other cellular or viral components. Following these processes by differential scanning calorimetry revealed four different transitions that correlated with structural transitions in the coat protein shells. Exit of scaffolding protein from the procapsid occurred reversibly and just above physiological temperature. Expansion of the procapsid lattice, which was exothermic, occurred after the release of scaffolding protein. Partial denaturation of coat subunits within the intact shell structure was detected prior to the major endothermic event. This major endotherm occurred above 80 degrees C and represents particle breakage and irreversible coat protein denaturation. The results indicate that the coat subunits are designed to form a metastable precursor lattice, which appears to be separated from the mature lattice by a kinetic barrier.

Acrylic Resins↗
PubMed1993

Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells.

The polymerization of protein subunits into precursor shells empty of DNA is a critical process in the assembly of double-stranded DNA viruses. For the well-characterized icosahedral procapsid of phage P22, coat and scaffolding protein subunits do not assemble separately but, upon mixing, copolymerize into double-shelled procapsids in vitro. The polymerization reaction displays the characteristics of a nucleation limited reaction: a paucity of intermediate assembly states, a critical concentration, and kinetics displaying a lag phase. Partially formed shell intermediates were directly visualized during the growth phase by electron microscopy of the reaction mixture. The morphology of these intermediates suggests that assembly is a highly directed process. The initial rate of this reaction depends on the fifth power of the coat subunit concentration and the second or third power of the scaffolding concentration, suggesting that pentamer of coat protein and dimers or trimers of scaffolding protein, respectively, participate in the rate-limiting step.

Bacteriophage P22↗
PubMed1993

Resource utilization in treatment of diabetic ketoacidosis in adults.

This study defines determinants of cost of treatment of diabetic ketoacidosis. A chart review for 92 cases of ketoacidosis from 1988 to 1992 in 40 females and 25 males (age range 18 to 81) showed a length of stay of 5.7 +/- 4.9 d. Length of stay did not correlate with the level of nursing care or any other component of the initial order set. Length of stay was shorter in cases managed by diabetologists. Length of stay was inversely proportional to the interval from arrival to the first shot of intermediate or long-acting insulin. Length of stay was longer in subjects with a positive bacterial culture (mean, 9.1 d) and was longer in subjects who arrived in the evening. There was a female predominance in total and recurrent cases of ketoacidosis. Female patients received fewer educational sessions than males. The grade of acidosis affected the duration of acidemia, but the grade of acidosis, APACHE scores, and admission lab values did not correlate with length of stay. The use of an intensive care unit (ICU) included more testing and expense without uniform clear benefit. Optimal transition from intensive to routine management includes resumption of long-acting insulins as soon as possible. Optimization of hospital care and reduction of incidence of ketoacidosis in females would have a marked effect on health care costs.

Adolescent↗
PubMed1993

Developing a restraint use policy for acute care.

Restraint use has been a recent focus of attention in long-term care facilities. The Joint Commission on Accreditation of Healthcare Organizations, the Commission on Accreditation of Rehabilitation Facilities, and the Food and Drug Administration have devoted attention to the prudent use of restraints. The authors address efforts of an acute care facility to comply with these regulations.

Aged↗
PubMed1993

Cloning, heterologous expression, and characterization of the Erysipelothrix rhusiopathiae DnaK protein.

The dnaK (hsp70) gene from the facultative intracellular pathogen Erysipelothrix rhusiopathiae was cloned by heterologous DNA hybridization of a genomic library using the Escherichia coli dnaK gene as a probe. A 3.2-kb fragment which encoded an 1,800-bp open reading frame was recovered. The deduced amino acid sequence of this open reading frame shares 56% identity with the E. coli DnaK protein. Expression of the encoded protein in E. coli by using the phage T7 promoter/polymerase system resulted in accumulation of a unique 65-kDa protein. Western blot (immunoblot) analysis of extracts from a recombinant E. coli strain using anti-E. coli DnaK polyclonal antibodies confirmed that the cloned gene encodes a DnaK homolog. The recombinant E. rhusiopathiae DnaK protein was purified to 80% homogeneity by ATP affinity chromatography. The purified material hydrolyzed ATP with a specific activity of 100 nmol min-1 mg of protein-1. Analysis of total protein extracts from E. rhusiopathiae indicates that DnaK is a highly expressed protein in this organism.

Adenosine Triphosphate↗
PubMed1993

Reform brings opportunities for restructuring hospital systems. Interview by Donald E. Johnson.

Donna Shalala, Health & Human Services secretary, approved Oregon's Medicaid reform plan which will reduce the cost of health care services to the poor while expanding access to the state's 120,000 uninsured residents. Health coverage will not be provided for Oregonians below the federal poverty level who currently don't receive any Medicaid benefits. Health Care Strategic Management publisher and editor Donald E. L. Johnson and John King, president and CEO of Legacy Health System, Portland, discuss the ramifications of the Oregon Plan and the opportunities it provides for Oregon hospital systems.

Health Care Rationing↗
PubMed1993

Safety and immunogenicity of a combined diphtheria, tetanus, pertussis and Haemophilus influenzae type b vaccine in young infants.

OBJECTIVE: To study the safety and immunogenicity of a combined diphtheria-tetanus-pertussis (DTP)-Haemophilus influenzae type b (HbOC) vaccine (TETRAMUNE) in infants as young as 2 months of age as compared to separate administration of DTP and HbOC. METHODS: Two-month-old infants were randomized to receive three doses 2 months apart of either DTP-HbOC as a single 0.5-mL injection or to receive 0.5 mL of DTP and HbOC concurrently in separate legs. Local and systemic adverse reactions were monitored within 72 hours of each immunization, and immunogenicity of each of the four vaccine components was measured. RESULTS: The incidence of both local and systemic adverse events following the tetravalent vaccine was similar to the incidence following separate vaccine administration. After three doses of vaccine, the response to each of the vaccine components was higher in the combined vaccine when compared to separate administration. In the case of the Haemophilus influenzae type b component, this enhancement was also seen after two doses. The response to the combined vaccine was consistent among the three lots tested as was the enhancement over separate administration. CONCLUSIONS: The DTP-HbOC vaccine was safe and immunogenic in young infants and was generally more immunogenic than separate vaccination with DTP and HbOC. The use of such a combined vaccine reduces the number of injections given to young infants by half and is an important step toward improving vaccine delivery.

Antibody Formation↗
PubMed1993

Ultrastructural hepatocellular features associated with severe hepatic lipidosis in cats.

In this study, we compared hepatic ultrastructure in healthy cats, in cats with severe hepatic lipidosis, and in cats with experimentally induced, chronic, extrahepatic bile duct occlusion. Ultrastructural features unique to the lipidosis syndrome included an apparent reduction in number of peroxisomes and alteration in their morphologic features. The quantity of endoplasmic reticulum, Golgi complexes, and lysosomes was subjectively reduced, and paucity of cytosolic glycogen was observed. Bile canaliculi appeared collapsed because of cytosolic distention with lipid. Mitochondria were reduced in number and were markedly pleomorphic. Cristae assumed a variety of shapes, lengths, and orientations. Ultrastructural features of bile duct occlusion were similar to those described in other species and differed from those in cats with hepatic lipidosis.

Animals↗
PubMed1992

Amino acid substitutions influencing intracellular protein folding pathways.

Though an increasing variety of chaperonins are emerging as important factors in directing polypeptide chain folding off the ribosome, the primary amino acid sequence remains the major determinant of final conformation. The ability to identify cytoplasmic folding intermediates in the formation of the tailspike endorhamnosidase of phage P22 has made it possible to isolate two classes of mutations influencing folding intermediates-temperature-sensitive folding mutations and global suppressors of tsf mutants. These and related amino acid substitutions in eukaryotic proteins are discussed in the context of inclusion body formation and problems in the recovery of correctly folded proteins.

Amino Acid Sequence↗
PubMed1992

The effects of nicotine plus haloperidol compared to nicotine only and placebo nicotine only in reducing tic severity and frequency in Tourette's disorder.

We studied the effects of chewing nicotine gum on tic frequency and severity in 10 patients with Tourette's disorder (TD) on haloperidol, versus 9 untreated TD patients; placebo gum was administered to 5 of these untreated patients. Videotapes of patients during a 2-hr period of 30 min baseline, 30 min gum chewing, and two 30-min postgum-chewing periods were utilized. For those TD patients on haloperidol, significant reductions occurred in tic frequency and severity during the gum-chewing and the two postgum-chewing periods. Nicotine gum alone caused a decrease in tic frequency only during gum-chewing and one postgum-chewing period, while placebo gum showed no effect. In this study, nicotine markedly potentiated haloperidol effects in treating TD, and showed lesser effects on TD when used alone.

Adolescent↗