PubMed Health⌕ Search

Biomedical subjects

D Kern

Publications and source records attributed to D Kern.

At least 37 records · Page 2Linked to original sources
PubMed1995

A kinetic analysis of the folding of human carbonic anhydrase II and its catalysis by cyclophilin.

The kinetics of unfolding and refolding of human carbonic anhydrase II (HCAII) and its catalysis by the peptidyl-prolyl-cis/trans-isomerase cyclophilin were investigated. HCAII contains 15 trans- and 2 cis-prolyl peptide bonds, and, when long-term denatured, virtually all unfolded molecules contain non-native prolyl isomers. In unfolding these molecules (Us) are produced slowly in a biphasic process reflecting the isomerization of several trans-prolines and of one cis-proline. In refolding, the rapid formation of an intermediate of the molten globule type is followed by several slow prolyl isomerizations, which determine the rate of reactivation. By a short 10-s incubation in 5.0 M guanidinium chloride at 2 degrees C, unfolded HCAII species with all prolines still in the native conformation (Uf) could be produced. Surprisingly, only a fraction of Uf refolds rapidly, but the other molecules refold slowly. Evidently, some prolyl peptide bonds isomerize early in refolding, at the stage of the molten globule and as a consequence, molecules with incorrect prolyl isomers are formed in competition with the productive folding of Uf. This fraction of slow-folding molecules is strongly increased when cyclophilin is present, because it accelerates the formation of non-native prolyl isomers as long as the molecules remain in the molten globule state. Later cyclophilin catalyzes the isomerization of these prolyl peptide bonds toward the native state, which are stabilized in their conformation by further folding to the native state. This catalysis is very efficient, because only prolines that are accessible in the molten globule are involved in this sequence of isomerization and reisomerization.

Amino Acid Isomerases↗
PubMed1995

Performance characteristics for the quantitation of plasma HIV-1 RNA using branched DNA signal amplification technology.

Highly sensitive assays that quantitate human immunodeficiency virus type 1 (HIV-1) RNA may be valuable for clinical research and the treatment of HIV-1-infected patients. In this study we evaluated the reproducibility and accuracy of the first-generation branched DNA (bDNA-1.0) signal amplification assay under conditions that are relevant to routine use in a clinical context. We show that the bDNA-1.0 assay was able to discern two- to three-fold changes in plasma HIV-1 RNA levels as significant. Reverse transcription coupled to polymerase chain reaction (RT-PCR) was less reproducible and required a 3.7- to 5.8-fold change in plasma HIV-1 RNA levels to be statistically significant. The accuracy of the bDNA-1.0 assay in RNA quantitation was not affected by HIV-1 genotypic variation or by the presence of hemoglobin, bilirubin, lipemia, or any of a dozen therapeutic drugs. Using the bDNA-1.0 assay, we show that HIV-1 RNA levels in plasma specimens were stable when stored at -80 degrees C and were able to withstand at least three freeze-thaw cycles without significant loss. We also examined the performance of an ultrasensitive bDNA assay with improvements to the signal amplification technology. The ultrasensitive bDNA assay displayed a quantitation limit of approximately 500 RNA Eq/ml, yet maintained a dynamic quantitation range up to 1.6 x 10(6) RNA Eq/ml. Like the bDNA-1.0 assay, the ultrasensitive bDNA assay was not affected by HIV-1 genotype variability.

Anticoagulants↗
PubMed1994

Implementing advance directives in the primary care setting.

BACKGROUND: Despite the potential benefits of advance directives, few patients complete them. This study examined whether barriers to advance decision making can be overcome via a combined educational and administrative intervention targeted at physicians. METHOD: The subjects consisted of all the internists (n = 6) at a primary care physician home care (HC) service and all the internists (n = 4) at a primary care nursing home (NH) service. Physicians were given a 5-week course on the law relating to advance directives. Administrative consent was obtained to permit physicians to spend additional time with patients to discuss advance directives. Physicians were asked to discuss advance directives with newly enrolled patients and to assist interested patients to complete directives. During the first 2 months of the trial, physicians did not approach any patients. Therefore, the study design was changed to include all active patients, and physicians received additional training that involved observing and leading discussions with their own patients. RESULTS: Physicians approached 74 of 356 competent HC patients, of whom 48 (65%) completed directives. All 42 competent NH patients were approached, and 38 (90%) completed directives. Most patients who completed a directive chose relatives as proxies. Most directed that life-sustaining treatment be withheld in the event they were permanently unconscious (HC, 81%; NH, 92%). Other common choices were to decline long-term mechanical ventilation (HC, 58%; NH, 79%), long-term artificial nutrition (HC, 44%; NH, 79%), and cardiopulmonary resuscitation (HC, 27%; NH, 66%). CONCLUSIONS: Physicians can overcome initial reluctance to integrate advance decision making into primary care provided to elderly patients. Teaching physicians about the law is not sufficient to change behavior; physicians also need practical experience discussing directives with patients. Our high patient response suggests that a physician-directed intervention is sufficient to achieve high rates of completing directives without additional, concomitant patient-directed intervention.

Advance Directives↗
PubMed1994

Crystallisation of the glycyl-tRNA synthetase from Thermus thermophilus and initial crystallographic data.

The glycyl-tRNA synthetase from Thermus thermophilus is a dimer of molecular mass 115 kDa, which has been crystallised using the vapour diffusion method from 5 to 7% polyethylene glycol 6000, 0.8 to 1.4 M NaCl at protein concentrations of 2 to 8 mg/ml. Nucleation is carried out at 4 degrees C and crystals are subsequently transferred to 15 degrees C to maximise growth. Crystals are truncated rhombohedra measuring on average 0.4 mm x 0.4 mm x 0.2 mm, which appear within a few days and reach full size in one to two months. GlyRS crystallises in two closely related space groups, P2(1)2(1)2(1) and C2,2,2(1), both with the same cell a = 125 A, b = 254 A, c = 104 A. Crystal packing in P2(1)2(1)2(1) is strongly C-centred. The crystals have VM = 3.6 A3/Da and a solvent content of 61%, with one dimer in the asymmetric unit in C2,2,2(1) and two dimers in P2(1)2(1)2(1). The best native data extend to 2.9 A in C2,2,2(1) and are 90.6% complete with an R-factor between symmetry-related reflections of 10.0%. The structure has been solved by multiple isomorphous replacement and model building is in progress.

Crystallization↗
PubMed1994

The ribosomal protein S8 from Thermus thermophilus VK1. Sequencing of the gene, overexpression of the protein in Escherichia coli and interaction with rRNA.

The gene of the ribosomal protein S8 from Thermus thermophilus VK1 has been isolated from a genomic library by hybridization of an oligonucleotide coding for the N-terminal amino acid sequence of the protein, amplified by PCR and sequenced. Nucleotide sequence reveals an open reading frame coding for a protein of 138 amino acid residues (M(r) 15,839). The codon usage shows that 94% of the codons possess G or C in the third position, and agrees with the preferential usage of codons of high G+C content in the bacteria of the genus Thermus. The amino acid sequence of the protein shows 48% identity with the protein from Escherichia coli. Ribosomal protein S8 from T. thermophilus has been expressed in E. coli under the control of the T7 promoter and purified to homogeneity by heat treatment of the extract followed by cation-exchange chromatography. Conditions were defined in which T. thermophilus protein S8 binds specifically an homologous 16S rRNA fragment containing the putative S8 binding site with an apparent association constant of 5 x 10(7) M-1. The overexpressed protein binds the rRNA with the same affinity as that extracted from T. thermophilus, indicating that the thermophilic protein is correctly folded in E. coli. The specificity of this binding is dependent on the ionic strength. The protein S8 from T. thermophilus recognizes the E. coli rRNA binding sites as efficiently as the S8 protein from E. coli. This result agrees with sequence comparisons of the S8 binding site on the small subunit rRNA from E. coli and from T. thermophilus, showing strong similarities in the regions involved in the interaction. It suggests that the structural features responsible for the recognition are conserved in the mesophilic and thermophilic eubacteria, despite structural peculiarities in the thermophilic partners conferring thermostability.

Amino Acid Sequence↗
PubMed1994

Reassessment of the putative chaperone function of prolyl-cis/trans-isomerases.

The folding of proteins can be assisted by two unrelated groups of helper molecules. Chaperones suppress non-productive side reactions by stoichiometric binding to folding intermediates, and folding enzymes catalyze slow rate-limiting steps of folding. We reinvestigated, whether peptidyl-prolyl-cis/trans-isomerases of the cyclophilin type act simultaneously as chaperones and as folding catalysts in the reactivation of human carbonic anhydrase II, as reported recently [Freskgård, P.-O. et al. (1992) Science 258, 466-468; Rinfret, A. et al. (1994) Biochemistry 33, 1668-1673]. No increase in the yield of native carbonic anhydrase-II could be detected in the presence of three different prolyl isomerases, when reactivation was followed by a sensitive assay for an extended time of 4 h. We conclude that the role of prolyl isomerases in the refolding of carbonic anhydrase can be explained solely by their isomerase activity. There is no need to invoke simultaneous functions as chaperones for these folding catalysts.

Amino Acid Isomerases↗
PubMed1994

Threonyl-tRNA synthetase from Thermus thermophilus: purification and some structural and kinetic properties.

Threonyl-tRNA synthetase (ThrRS) has been isolated from an extreme thermophile Thermus thermophilus strain HB8. The enzyme was purified to electrophoretic homogeneity by combinations of column chromatographies on DEAE-Sepharose, S-Sepharose, ACA-44 Ultrogel and HA-Ultrogel. Seventeen mg of purified enzyme were obtained from 1 kg of biomass. In parallel, purified aspartyl- and phenylalanyl-tRNA synthetases were obtained. The purified ThrRS is composed of two identical subunits with a molecular mass of about 77,000 (virtually the same as E coli ThrRS). The N-terminal sequence has been determined. The homology between the first 45 amino acid residues of ThrRS from T thermophilus and E coli is about 29%. A comparative study of tRNA(Thr) charging by ThrRS from E coli and T thermophilus reveals a similar efficiency of the reaction in both homologous systems. This efficiency remains unchanged for aminoacylation of tRNA(Thr) from T thermophilus by the heterologous ThrRS from E coli, but decreases 700 times for aminoacylation of E coli tRNA(Thr) by ThrRS from T thermophilus.

Amino Acid Sequence↗
PubMed1994

Impact of special care unit for patients with advanced Alzheimer's disease on patients' discomfort and costs.

OBJECTIVE: To compare outcomes in patients with the clinical diagnosis of probable dementia of the Alzheimer type (DAT) cared for in a Dementia Special Care Unit (DSCU) with those in traditional long-term care (TLTC). DESIGN: Two-year prospective cohort study. SETTING: Two Veterans Administration Hospitals. The DSCU concentrated on assuring patients' comfort instead of promoting maximal survival; in some patients this excluded transfer to acute medical settings, the use of antibiotics, and tube feeding. MEASUREMENTS: Data were collected regarding disease severity, patient discomfort, use of medical resources, and mortality rate. RESULTS: Patients at both settings were similar on baseline measures, and most were severely demented. The monthly levels of observed discomfort were lower in DSCU than in TLTC patients. The costs of medications, radiology, and laboratory procedures were lower in DSCU than in TLTC patients. DSCU patients were also transferred less frequently to an acute medical setting. The average 3-month cost for a DSCU patient was $1477 less than the cost of care for a TLTC patient. However, DSCU patients with lower severity of DAT had a higher mortality rate then TLTC patients. CONCLUSIONS: These results suggest that management of patients with advanced DAT on a DSCU using a palliative care philosophy may result in less patient discomfort and lower costs than management on a TLTC.

Aged↗
PubMed1993

Sequence, overproduction and crystallization of aspartyl-tRNA synthetase from Thermus thermophilus. Implications for the structure of prokaryotic aspartyl-tRNA synthetases.

The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strain VK-1 and HB8, have been cloned and sequenced. Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl-tRNA synthetases. This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones. When its gene is under the control of the tac promoter of the expression vector pKK223-3, the protein is efficiently overexpressed as a thermostable protein in E. coli. It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography. Single crystals of the pure protein, diffracting at least to 2.2 A resolution (space group P2(1)2(1)2(1), a = 61.4 A, b = 156.1 A, c = 177.3 A) are routinely obtained. The same crystals have previously been described as crystals of threonyl-tRNA synthetase [1].

Amino Acid Sequence↗
PubMed1993

The cis/trans interconversion of the calcium regulating hormone calcitonin is catalyzed by cyclophilin.

The cytosolic peptidyl-prolyl cis/trans isomerase cyclophilin from pig kidney can accelerate catalytically the cis/trans isomerization of prolyl peptide bonds. One- and two-dimensional 1H NMR spectroscopy was used to prove that the polypeptide hormone calcitonin is a substrate for cyclophilin. Isomerization of only one of the two prolyl peptide bonds is catalyzed significantly. The efficiency of catalysis was calculated by lineshape analysis and NOESY spectroscopy. Cyclosporin A completely blocks the effect of the enzyme on the conformational dynamics of the polypeptide.

Amino Acid Isomerases↗
PubMed1993

Kinetics of folding and association of differently glycosylated variants of invertase from Saccharomyces cerevisiae.

A core-glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well-defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high-mannose-glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene. Renaturation of core-glycosylated invertase upon dilution from guanidinium-chloride solutions follows a unibimolecular reaction scheme with consecutive first-order subunit folding and second-order association reactions. The rate constant of the rate-limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 +/- 0.4 x 10(-3) s-1 at 20 degrees C; it is characterized by an activation enthalpy of delta H++ = 65 kJ/mol. The reaction is not catalyzed by peptidyl-prolyl cis-trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size-exclusion high-pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 x 10(3) M-1 s-1 to 7 x 10(4) M-1 s-1 between 5 and 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Reactivators↗
PubMed1993

Cognitive function and spirometry performance in the elderly.

A total of 65 ambulatory subjects over the age of 65 yr were studied to determine if mild impairment in cognitive function precludes reliable spirometric measures in the aged. Standardized questionnaires were used to obtain information on demographics, cigarette smoking, respiratory symptoms, and physician-diagnosed lung disease. Each subject performed several simple standardized tasks of cognitive function and underwent spirometric testing. A total of 36 women and 29 men participated. The mean age for the group was 74.9 +/- 5.6 yr; most were nonsmokers (never smokers, n = 28; former smokers, n = 29; and current smokers, n = 8). Of the 65 participants, 8 (12.3%) individuals were unable to perform at least three ATS-acceptable FVC maneuvers after suitable demonstration. These subjects were similar to the 57 subjects able to perform three acceptable maneuvers, except for worse scores on both the symbol-digit modalities test (23.3 +/- 3.6 versus 31.6 +/- 10.5, p < 0.001) and the trail-making test, Part B (244.3 +/- 87.1 versus 160.4 +/- 71.8, p < 0.01). Of the 57 subjects able to perform spirometry, 18 (31.6%) failed to meet ATS reproducibility criteria for FEV1, FVC, or both. Cognitive impairment was not associated with the ability to achieve reproducible measures. These results suggest that the vast majority of older subjects can perform reliable spirometry; those elderly unable to perform spirometry may have impairment in cognitive function requiring further evaluation.

Aged↗
PubMed1992

Kinetics of 1,25-dihydroxyvitamin D metabolism in the aging rat.

To determine whether postmaturational aging influences the kinetics of 1,25-dihydroxyvitamin D [1,25(OH)2D] metabolism in the rat, we measured the metabolic clearance (MCR) and production (PR) rates of 1,25(OH)2D in 6-, 12-, 18-, and 24-mo-old Fischer 344 rats using the constant infusion method. Plasma calcium, phosphorus, and parathyroid hormone (PTH), urinary calcium and phosphorus, and glomerular filtration rate (GFR) were also measured. MCR and PR increased 57 and 91%, respectively (when expressed per rat), and 32 and 39%, respectively (when expressed per kg body wt), between 6 and 24 mo of age, with the greatest increase occurring between 18 and 24 mo. The plasma concentration of 1,25(OH)2D remained unchanged. Plasma PTH, when compared with 6-mo-old animals, was significantly elevated at 18 mo (147%) and even higher at 24 mo (240%). GFR (51Cr-labeled EDTA clearance) remained unchanged through 18 mo but tended to be reduced at 24 mo. Plasma phosphorus tended to decrease with age, whereas plasma calcium, urinary calcium, and urinary phosphorus did not differ among the age groups. These data indicate that both clearance and production of 1,25(OH)2D increase with postmaturational aging in the rat. They also suggest that the early age-related rise in plasma PTH is neither a consequence of low plasma calcium nor of renal insufficiency.

Aging↗
PubMed1992

An exploration of the variables involved when instituting a do-not-resuscitate order for patients undergoing bone marrow transplantation.

Written do-not-resuscitate (DNR) orders can facilitate the transition from aggressive care to supportive care in patients undergoing bone marrow transplantation who are critically ill. The DNR decision often is complicated by clinical and ethical problems. To assist nurses in analyzing these complex situations, a retrospective review was conducted on medical records of 40 patients who died on an eight-bed bone marrow transplant unit in which a formal DNR policy was in place. A review of specific variables for patients designated DNR and non-DNR showed that the groups did not vary in age, diagnosis, disease, or type of transplant. The non-DNR group developed life-threatening complications earlier in their transplant course, whereas multisystem failure was the common factor among the patients with DNR designation. The few ethical problems documented in the medical records did not appear to reflect the complexity of the DNR decision.

Adolescent↗
PubMed1991

Isolation and characterization of active N-terminal truncated apo- and holoenzyme of mammalian liver tyrosine aminotransferase.

Limited proteolysis was used to probe the structure of the apo- and holoenzyme of rat liver tyrosine aminotransferase. Both were subjected to trypsinolysis and the major fragments were isolated and characterized. Trypsin cleaves the apoenzyme after residues Arg57, Lys64, and Lys71 and the holoenzyme after Arg37 and Lys38. The difference in the accessibility of the enzyme deprived or associated with pyridoxal 5'-phosphate reflects two distinct conformations. The activity, the affinity for the ligands and the thermostability of the purified truncated enzyme forms are similar to those of the native apo- and holoenzyme. A model for the domain structure of mammalian tyrosine aminotransferase and a mechanism for its rapid turnover are proposed.

Amino Acid Sequence↗