Biomedical subjects

J Carey

Publications and source records attributed to J Carey.

At least 37 records · Page 2Linked to original sources

PubMed1999

Role of heme in structural organization of cytochrome c probed by semisynthesis.

The heme prosthetic group of cytochrome c is covalently attached to the protein through thioether bonds to two cysteine side chains. The role of covalent heme attachment to cytochrome c is not understood, and most heme proteins bind the prosthetic group by iron ion ligation and tertiary interactions only. A two-armed attachment seems redundant if the role of covalent connection is to limit heme group orientation or to decouple heme affinity from redox potential. These considerations suggested that one role for covalent attachment of the rigid planar heme might be in organizing the cytochrome c protein structure. Indeed, porphyrin cytochrome c (in which the heme iron ion has been removed) is substantially more ordered than apocytochrome c, having characteristics consistent with a molten globule state. To assess the importance of planar rigidity in ordering this protein, semisynthesis was used to substitute porphyrin by two hydrophobic surrogates, one based on biphenyl and the other on phenanthrene, which have different degrees of planarity and rigidity. The expected two-armed covalent attachment of each surrogate was confirmed in the protein products by a variety of methods including mass spectrometry and NMR. Despite being only about half the size of the porphyrin macrocycle, and lacking any possibility for ligation or polar group interactions with the surrounding protein, the two surrogates confer helix contents that are comparable to that of the molten globule formed by porphyrin cytochrome c under similar solution conditions. The pH titrations of the derivatives monitored by circular dichroism exhibit reversible, bell-shaped folding and unfolding transitions, implying that charge group interactions in the protein are involved in stabilizing the helical structures formed. The thermal transitions of the two derivatives at neutral pH are cooperative, with similar midpoints. The similarity of helical content and structural stability in the two derivatives indicates that the increase in conformational freedom by the biphenyl surrogate does not substantially reduce protein structural stability. The similarity of the two derivatives to porphyrin cytochrome c suggests that the common feature among the three covalently attached groups-their hydrophobicity-is by far the dominant factor in organizing stable structures in the protein.

Biphenyl Compounds↗

PubMed1999

Protein purification with vapor-phase carbon dioxide.

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.

Alkaline Phosphatase↗

PubMed1999

Structural organization in peptide fragments of cytochrome c by heme binding.

Prosthetic groups are often important structural organizers of proteins as well as essential functional components. Insertion of prosthetic groups is usually spontaneous, and implies an apoprotein that is partially preorganized to provide a recognition surface for specific binding. Cytochrome c is distinguished by having its heme attached by a dedicated heme lyase through thioether links to cysteine side-chains, and the apoprotein shows no evidence of preorganization under physiological conditions. Nevertheless, addition of heme to two short fragments of cytochrome c enhances helical structure substantially (from approximately 8% to approximately 22%), an effect that depends on iron ligation but not thioether linkage. The helical segments in the corresponding parts of the native holoprotein have little contact surface with heme, implying that the increased helical structure in the fragment complex may depend on tertiary interactions. The absence of the intervening polypeptide chain suggests that the complex represents a relatively independent folded subdomain.

Amino Acid Sequence↗

PubMed1999

Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor.

A mutant tryptophan repressor (TrpR) protein containing the substitution of phenylalanine for leucine 75 has been isolated following a genetic screen for temperature-sensitive mutations. Two-dimensional (2D) 1H NMR spectra indicate an overall very similar fold for the purified mutant and wild-type proteins. Circular dichroism spectropolarimetry indicates an increased helix content relative to the wild-type protein, and a slightly higher urea denaturation midpoint for the mutant protein, although there is no difference in thermal stability. Fluorescence spectra indicate a more buried environment for one or both tryptophan residues in the mutant protein. The rate of proton-deuterium exchange-out for the resolved indole ring protons of the two tryptophan residues was quantified from NMR spectra of mutant and wild-type proteins and found to be approximately 50% faster in the wild-type protein. The mutant protein binds the corepressor l-tryptophan (l-Trp) approximately ten times more weakly than does the wild-type protein, but in l-Trp excess its DNA-binding affinity is only two to fivefold weaker. Taken together the results imply that, despite its conservative chemical character and surface location at the C terminus of helix one in the helix-turn-helix DNA recognition motif, this mutational change confers long-range effects on the dynamics of the protein's secondary and tertiary structure without substantially altering its fold, and with relatively minor effects on protein function.

Amides↗

PubMed1999

Cooperative folding units of escherichia coli tryptophan repressor.

A previously published computational procedure was used to identify cooperative folding units within tryptophan repressor. The theoretical results predict the existence of distinct stable substructures in the protein chain for the monomer and the dimer. The predictions were compared with experimental data on structure and folding of the repressor and its proteolytic fragments and show excellent agreement for the dimeric form of the protein. The results suggest that the monomer, the structure of which is currently unknown, is likely to have a structure different from the one it has within the context of the highly intertwined dimer. Application of this method to the repressor monomer represents an extension of the computations into the realm of evaluating hypothetical structures such as those produced by threading.

Apoproteins↗

PubMed1999

Contribution of meniscal extrusion and cartilage loss to joint space narrowing in osteoarthritis.

OBJECTIVE: This study was undertaken to determine the contribution of meniscal extrusion and cartilage loss to joint space narrowing on conventional radiographs by correlation with magnetic resonance imaging (MRI). SUBJECTS AND METHODS: Sixty-two consecutive patients, 32 patients with osteoarthritis and 30 without osteoarthritis, over the age of 60 years that were referred for both radiographic and MRI of the knee were included in the study. In each case, relative joint space narrowing on conventional AP radiographs was assessed utilizing the Kellgren-Lawrence scoring system. Subsequently, the degree of meniscal extrusion and the integrity of articular cartilage were evaluated from MR in the same patients. RESULTS: Each of 30 patients with normal joint space (Kellgren Grade 0) were noted to have normal articular cartilage, grade 1 meniscal extrusion was identified in only three of these patients. In comparison, meniscal extrusion was identified in all 32 patients with joint space narrowing (Kellgren Grades 1-4). Definite thinning or loss of articular cartilage was identified in only 15 of the 32 cases. In 17 patients with radiographic joint space narrowing (Kellgren Grades 1-3) and meniscal extrusion, no loss of articular cartilage was observed. A statistically significant correlation (P<0.001) was observed between Kellgren Grade and degree of meniscal extrusion and cartilage thinning on MRI. CONCLUSION: Conventional radiographs are an unreliable method of evaluating for articular cartilage loss in patients with early osteoarthritis. Initial joint space narrowing on conventional radiographs is secondary to meniscal extrusion rather than thinning of articular cartilage in most cases.

Aged↗

PubMed1999

Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis.

Cathepsin K, a lysosomal cysteine protease critical for bone remodeling by osteoclasts, was recently identified as the deficient enzyme causing pycnodysostosis, an autosomal recessive osteosclerotic skeletal dysplasia. To investigate the nature of molecular lesions causing this disease, mutations in the cathepsin K gene from eight families were determined, identifying seven novel mutations (K52X, G79E, Q190X, Y212C, A277E, A277V, and R312G). Expression of the first pro region missense mutation in a cysteine protease, G79E, in Pichia pastoris resulted in an unstable precursor protein, consistent with misfolding of the proenzyme. Expression of five mature region missense defects revealed that G146R, A277E, A277V, and R312G precursors were unstable, and no mature proteins or protease activity were detected. The Y212C precursor was activated to its mature form in a manner similar to that of the wild-type cathepsin K. The mature Y212C enzyme retained its dipeptide substrate specificity and gelatinolytic activity, but it had markedly decreased activity toward type I collagen and a cathepsin K-specific tripeptide substrate, indicating that it was unable to bind collagen triple helix. These studies demonstrated the molecular heterogeneity of mutations causing pycnodysostosis, indicated that pro region conformation directs proper folding of the proenzyme, and suggested that the cathepsin K active site contains a critical collagen-binding domain.

Cathepsin K↗

PubMed1999

Validity of three measures of severity of AIDS for use in health services research studies.

Policy makers and hospital managers often use severity adjustments as a control for patient mix differences when evaluating outcomes of care. Unfortunately, few indices are carefully examined and therefore the evaluations based on these methods of severity adjustment are suspect. This paper examines the accuracy of three indices for measuring the severity of illness of AIDS patients. We examine the Diagnosis-Based Severity Index (DBSI), a modified version of DBSI referred to as MDBSI and the Composite Laboratory Index (CLI) in predicting survival of AIDS patients at one medical centre. We analysed the correlation between indices and months of survival. We also examined the percentage of variance in survival months explained by each index separately and together. Finally, we used survival analysis to examine whether DBSI classifies patients in groups with distinct patterns of survival. Only patients who had died were included in the analysis so that information on the patients' full course of illness was available. Of the 91 patients abstracted, 81 cases had date of AIDS, date of death, and the CLI. These 81 cases were the focus of the analysis. Both CLI and DBSI were predictive of months of survival but were not correlated to each other. Predictions of months of survival were improved if both indices were used together rather than separately. Survival analysis confirmed that patients classified by DBSI had distinctly different survival patterns. Each index measures different aspects of the severity of the patient's condition and when possible both indices should be used together. When laboratory data are not available, e.g. in Medicaid administrative files, the use of DBSI may be reasonable.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Water molecules in DNA recognition I: hydration lifetimes of trp operator DNA in solution measured by NMR spectroscopy.

The present NMR study investigates the residence times of the hydration water molecules associated with uncomplexed trp operator DNA in solution by measuring intermolecular nuclear Overhauser effects (NOE) between water and DNA protons, and the nuclear magnetic relaxation dispersion (NMRD) of the water 2H and 17O resonances. Both methods indicate that the hydration water molecules exchange with bulk water on the sub-nanosecond time scale at 4 degreesC. No evidence was obtained for water molecules bound with longer residence times. In particular, the water molecules at the sites of interfacial hydration in the trp repressor/operator complex do not seem kinetically stabilized in the uncomplexed DNA. Analysis of the crystal structures of two different trp repressor/operator complexes shows very similar structural environments for the water molecules mediating specific contacts between the protein and the DNA, whereas much larger variations are observed for the location of corresponding water molecules detected in the crystal structure of an uncomplexed trp operator DNA duplex. Therefore, it appears unlikely that the hydration characteristics of the uncomplexed DNA target would be a major determinant of trp repressor/operator recognition.

Bacterial Proteins↗

PubMed1998

Biochemical characterization of WrbA, founding member of a new family of multimeric flavodoxin-like proteins.

The protein WrbA had been identified as an Escherichia coli stationary-phase protein that copurified and coimmunoprecipitated with the tryptophan repressor. Sequences homologous to WrbA have been reported in several species of yeast and plants. We previously showed that this new family of proteins displays low but structurally significant sequence similarity with flavodoxins and that its members are predicted to share the alpha/beta core of the flavodoxin fold but with a short conserved insertion unique to the new family, which could account for reports that some family members may be dimeric in solution. The general sequence similarity to flavodoxins suggests that the members of the new family might bind FMN, but their wide evolutionary distribution indicates that, unlike the flavodoxins, these proteins may be ubiquitous. In this paper, we report the purification and biochemical characterization of WrbA, demonstrating that the protein binds FMN specifically and is a multimer in solution. The FMN binding constant is weaker than for many flavodoxins, being approximately 2 microM at 25 degreesC in 0. 1 mM sodium phosphate, pH 7.2. The protein participates in a dimer-tetramer equilibrium over a wide range of solution conditions, with a midpoint at approximately 1.4 microM. One FMN binds per monomer and has no apparent effect on the multimerization equilibrium. WrbA has no effect on the affinity or mode of DNA binding by the tryptophan repressor; thus, its physiological role remains unclear. Although many proteins with flavodoxin-like domains are known to be multimers, WrbA is apparently the first characterized case in which multimerization is associated directly with the flavodoxin-like domain itself.

Bacterial Proteins↗

PubMed1998

MRI of pediatric growth plate injury: correlation with plain film radiographs and clinical outcome.

PURPOSE: To evaluate pediatric growth plate injuries with conventional radiographs and magnetic resonance imaging (MRI). To review potential clinical impact of MRI on subsequent patient management and outcome. METHODS: Fourteen patients with known or suspected growth plate injury were studied. Each patient underwent imaging by conventional radiography and MRI within 2 weeks of injury. Findings on conventional radiographs and on MR images were compared and then correlated with subsequent management and outcome at a mean of 12 months. RESULTS: Direct visualization of cartilage afforded by MRI improved evaluation of growth plate injury in each case. MRI changed Salter Harris classification or staging in 2 of 9 patients with fractures visualized on conventional radiographs, allowed the detection of radiographically occult fractures in 5 of 14 cases, and resulted in a physical change in management in 5 of the 14 patients studied. CONCLUSION: MRI has an important role in the evaluation of acute pediatric growth plate injury, particularly when diagnostic uncertainty persists following the evaluation of conventional radiographs. MRI allows detection of occult fractures, may alter Salter Harris staging, and in the reported study it frequently resulted in a change in patient management.

Adolescent↗

PubMed1998

Randomized trial of itraconazole oral solution for oropharyngeal candidiasis in HIV/AIDS patients.

PURPOSE: Oropharyngeal candidasis (thrush) is the most common opportunistic infection in individuals who are positive for the human immunodeficiency virus (HIV) and those who have progressed to AIDS. Itraconazole has a broad in vitro spectrum of activity, including a wide variety of Candida species. Our study determined the relative efficacy of a new oral solution formulation of itraconazole and fluconazole tablets in the treatment of oropharyngeal candidiasis. PATIENTS AND METHODS: This was a prospective randomized, third-party-blind, multicenter trial conducted at 12 centers in the United States. One hundred seventy-nine HIV-positive patients with mycologically documented oropharyngeal candidiasis were treated with itraconazole oral solution 200 mg/ day for 7 or 14 days, or fluconazole tablets 100 mg/day for 14 days. Severity of disease was scored clinically before treatment and at clinical evaluations on days 3, 7, 14, 21, 35, and 42. Semi-quantitative cultures of mouth washings were also obtained on these days. RESULTS: Both 14-day and 7-day regimens of itraconazole oral solution were equivalent to fluconazole for most efficacy parameters. The clinical response rate was 97% after 14 days of itraconazole and 87% after 14 days of fluconazole. Itraconazole oral solution given for 7 days was also equivalent to fluconazole treatment for 14 days. Approximately one half of patients in all three groups relapsed by 1 month after completion of treatment. There were few adverse reactions to either drug. CONCLUSION: Itraconazole oral solution is well tolerated and offers an alternative at least as effective as fluconazole in the treatment of oropharyngeal candidiasis.

AIDS-Related Opportunistic Infections↗

PubMed1998

Isolated scaphotrapeziotrapezoid osteoarthritis: a possible radiographic marker of chronic scapholunate ligament disruption.

This study was undertaken to describe 'isolated' osteoarthritis of the scaphotrapeziotrapezoid articulation in six patients with chronic scapholunate ligament disruption. Each patient included for study had scapholunate ligament disruption with secondary dorsal intercalated segmental instability. Secondary volar tilt of the scaphoid appeared to be limited in each case by impaction and development of osteoarthritis at the scaphotrapeziotrapezoid articulation. Isolated osteoarthritis at the scaphotrapeziotrapezoid articulation may accompany chronic scapholunate ligament disruption, its identification should trigger a search for associated scapholunate ligament disruption in the absence of a known systemic arthropathy.

Chronic Disease↗

PubMed1997

Molecular and biological constraints on ligand-binding affinity and specificity.

Interactions between biological macromolecules have characteristic values of affinity and specificity that are set according to the biological function that is served by the interaction in the organism. Here we examine the molecular mechanisms that are used to achieve the required values of affinity and specificity in various biological systems.

DNA↗

PubMed1997

Long-term and high-temperature storage of supercritically-processed microparticulate protein powders.

PURPOSE: The long-term and high-temperature storage of dry, micron-sized particles of lysozyme, trypsin, and insulin was investigated. Subsequent to using supercritical carbon dioxide as an antisolvent to induce their precipitation from a dimethylsulfoxide solution, protein microparticles were stored in sealed containers at -25, -15, 0, 3, 20, 22, and 60 degrees C. The purpose of this study was to investigate the suitability of supercritical antisolvent precipitation as a finishing step in protein processing. METHODS: Karl Fisher titrations were used to determine the residual moisture content of commercial and supercritically-processed protein powders. The secondary structure of the dry protein particles was determined periodically during storage using Raman spectroscopy. The proteins were also redissolved periodically in aqueous buffers and assayed spectrophotometrically for biological activity and by circular dichroism for structural conformation in solution. RESULTS: Amide I band Raman spectra indicate that the secondary structure of the protein particles, while perturbed from that of the solution state, remained constant in time, regardless of the storage temperature. The recoverable biological activity upon reconstitution for the supercritically-processed lysozyme and trypsin microparticles was also preserved and found to be independent of storage temperature. Far UV circular dichroism spectra support the bioactivity assays and further suggest that adverse structural changes, with potential to hinder renaturation upon redissolution, do not take place during storage. CONCLUSIONS: The present study suggests that protein precipitation using supercritical fluids may yield particles suitable for long-term storage at ambient conditions.

Animals↗

PubMed1997

Solution structure of the DNA-binding domain and model for the complex of multifunctional hexameric arginine repressor with DNA.

The structure of the monomeric DNA-binding domain of the Escherichia coli arginine repressor, ArgR, determined by NMR spectroscopy, shows structural homology to the winged helix-turn-helix (wHTH) family, a motif found in a diverse class of proteins including both gene regulators and gene organizers from prokaryotes and eukaryotes. Biochemical data on DNA binding by intact ArgR are used as constraints to position the domain on its DNA target and to derive a model for the hexamer-DNA complex using the known structure of the L-arginine-binding domain. The structural independence of the wHTH fold may be important for multimeric DNA-binding proteins that contact extended DNA regions with imperfect match to consensus sequences, a feature of many wHTH-domain proteins.

Amino Acid Sequence↗

PubMed1997

A three-step PCR protocol for construction of chimeric proteins.

A general method is described for creating chimeric proteins by transposition of subdomain-sized gene fragments. The method uses three sequential PCR steps to circumvent several limitations inherent in transposition of small insertions and has been optimized to avoid purification of intermediate products. The sequence-independence of the method permits flexibility in the choice of host molecule and insertion site.

Bacterial Proteins↗

PubMed1997

A comparison of whole-body turboSTIR MR imaging and planar 99mTc-methylene diphosphonate scintigraphy in the examination of patients with suspected skeletal metastases.

OBJECTIVE: This study was undertaken to compare whole-body turbo short inversion time inversion recovery MR imaging and 99mTc-methylene diphosphonate planar scintigraphy in the examination of patients with suspected skeletal metastases. SUBJECTS AND METHODS: Twenty-five patients with known or suspected skeletal metastatic disease underwent both whole-body turbo short inversion time inversion recovery MR imaging and whole-body 99mTc-methylene diphosphonate scintigraphy. RESULTS: MR imaging revealed metastases at 57 of 175 possible sites (sensitivity, 96.5%, specificity, 100%; positive predictive value, 100%). Scintigraphy revealed metastases at 43 of 175 possible sites (sensitivity, 72%; specificity, 98%; positive predictive value, 95%) (McNemar test, 0.01; p = .016). Discrepancies in skeletal evaluation by whole-body MR imaging and scintigraphy were observed in six (24%) of 25 patients. Soft-tissue abnormalities were identified in 13 (52%) of 25 patients with MR imaging alone. CONCLUSION: Preliminary results suggest that whole-body MR imaging is an effective method of examining patients with suspected skeletal metastases, with better sensitivity than conventional planar 99mTc-methylene diphosphonate scintigraphy.

Adult↗