Challenges comparing functional limitations in rheumatoid arthritis and ankylosing spondylitis.

Whether physical functioning in patients with rheumatoid arthritis (RA) differs from that in patients with ankylosing spondylitis (AS) is presently uncertain. Such a comparison poses challenges, not only because the two diseases differ in the domains of functioning affected, but also because of the different instruments used to measure functional limitations. Limiting our analysis to studies using similar self-report questionnaires, we examined published observational studies of unselected cohorts of patients with RA and patients with AS to compare and contrast the severity of functional limitations. Available studies from a few direct comparisons, and mostly indirect comparisons, suggested that patients with RA are generally more severely limited in physical functioning throughout the disease course than patients with AS. Since most studies did not adjust adequately for potentially important confounders, such as age, gender, comorbidity, and disease duration, reported differences in functional disability between patients with RA and patients with AS must be interpreted cautiously.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

A polypeptide chain-refolding event occurs in the Gly82 variant of yeast iso-1-cytochrome c.

The replacement of Phe82 in yeast iso-1-cytochrome c by a glycine residue substantially alters both the tertiary structure and electron transfer properties of this protein. The largest structural change involves a polypeptide chain refolding of residues 79 through 85. Refolding places glycines 82, 83 and 84 immediately adjacent to the plane of the heme group in a spatial positioning comparable to that of the phenyl ring of Phe82 in the wild-type protein. Despite this perturbation in structure, solvent accessibility computations show that heme solvent exposure has not increased in the Gly82 variant protein. However, refolding does result in the introduction of a number of polar groups into the hydrophobic heme pocket. This appears to be responsible for the decreased reduction potential of the heme in this protein. The present study, along with that of the Ser82 variant protein (Louie et al., 1988b), clearly establishes the link between dielectric constant within the heme crevice and reduction potential. The further anomalously low electron transfer activity of the Gly82 variant protein would appear to arise from two factors. First, the polypeptide chain medium now adjacent to the heme is unable to facilitate electron transfer in a manner similar to that of the aromatic side-chain of Phe82. Second, polypeptide chain refolding significantly alters the surface contour of the Gly82 protein rendering it less suitable to interact with the corresponding complementary surfaces of redox partners. Our data support the conclusion that Phe82 plays a number of roles in the electron transfer process mediated by yeast iso-1-cytochrome c. These include the maintenance of the heme environment, provision of an optimal medium along the path of electron transfer and formation of interactions at the contact interface in complexes with redox partners.

High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c.

The structure of yeast iso-1-cytochrome c has been refined against X-ray diffraction data to a nominal resolution of 1.23 A. The atomic model contains 893 protein atoms, as well as 116 water molecules and one sulfate anion. Also included in the refinement are 886 hydrogen atoms belonging to the protein molecule. The crystallographic R-factor is 0.192 for the 12,513 reflections with F greater than or equal to 3 sigma (F) in the resolution range 6.0 to 1.23 A. Co-ordinate accuracy is estimated to be better than 0.18 A. The iso-1-cytochrome c molecule has the typical cytochrome c fold, with the polypeptide chain organized into a series of alpha-helices and reverse turns that serve to envelop the heme prosthetic group in a hydrophobic pocket. Inspection of the conformations of helices in the molecule shows that the local environments of the helices, in particular the presence of intrahelical threonine residues, cause distortions from ideal alpha-helical geometry. Analysis of the internal mobility of iso-1-cytochrome c, based on refined crystallographic temperature factors, shows that the most rigid parts of the molecule are those that are closely associated with the heme group. The degree of saturation of hydrogen-bonding potential is high, with 90% of all polar atoms found to participate in hydrogen bonding. The geometry of intramolecular hydrogen bonds is typical of that observed in other high-resolution protein structures. The 116 water molecules present in the model represent about 41% of those expected to be present in the asymmetric unit. The majority of the water molecules are organized into a small number of hydrogen-bonding networks that are anchored to the protein surface. Comparison of the structure of yeast iso-1-cytochrome c with those of tuna and rice cytochromes c shows that these three molecules have very high structural similarity, with the atomic packing in the heme crevice region being particularly highly conserved. Large conformational differences that are observed between these cytochromes c can be explained by amino acid substitutions. Additional subtle differences in the positioning of the side-chains of several highly conserved residues are also observed and occur due to unique features in the local environments of each cytochrome c molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Salt, phosphate and the Bohr effect at the hemoglobin beta chain C terminus studied by hydrogen exchange.

Hydrogen exchange experiments using functional labeling and fragment separation methods were performed to study interactions at the C terminus of the hemoglobin beta subunit that contribute to the phosphate effect and the Bohr effect. The results show that the H-exchange behavior of several peptide NH at the beta chain C terminus is determined by a transient, concerted unfolding reaction involving five or more residues, from the C-terminal His146 beta through at least Ala142 beta, and that H-exchange rate can be used to measure the stabilization free energy of interactions, both individually and collectively, at this locus. In deoxy hemoglobin at pH 7.4 and 0 degrees C, the removal of 2,3-diphosphoglycerate (DPG) or pyrophosphate (loss of a salt to His143 beta) speeds the exchange of the beta chain C-terminal peptide NH protons by 2.5-fold (at high salt), indicating a destabilization of the C-terminal segment by 0.5 kcal of free energy. Loss of the His146 beta 1 to Asp94 beta 1 salt link speeds all these protons by 6.3-fold, indicating a bond stabilization free energy of 1.0 kcal. When both these salt links are removed together, the effect is found to be strictly additive; all the protons exchange faster by 16-fold indicating a loss of 1.5 kcal in stabilization free energy. Added salt is slightly destabilizing when DPG is present but provides some increased stability, in the 0.2 kcal range, when DPG is absent. The total allosteric stabilization energy at each beta chain C terminus in deoxy hemoglobin under these conditions is measured to be 3.8 kcal (pH 7.4, 0 degrees C, with DPG). In oxy hemoglobin at pH 7.4 and 0 degrees C, stability at the beta chain C terminus is essentially independent of salt concentration, and the NES modification, which in deoxy hemoglobin blocks the His146 beta to Asp94 beta salt link, has no destabilizing effect, either at high or low salt. These results appear to show that the His146 beta salt link, which participates importantly in the alkaline Bohr effect, does not reform to Asp94 beta or to any other salt link acceptor in a stable way in oxy hemoglobin at low or high salt conditions.

Allosteric energy at the hemoglobin beta chain C terminus studied by hydrogen exchange.

When hemoglobin switches from the deoxy (T) to the liganded (R) form, several of its peptide group NH experience a great increase in their rate of exchange with water. Selective labeling and fragment isolation experiments identify some of the sensitive protons as three to four near-neighbor H-bonded peptide NH placed between Ala140 beta and the C-terminal His146 beta residue. These NH have differing solvent accessibilities, yet all exchange at about the same rate, and they maintain a common rate in the face of modifications that change their exchange rate over a 1000-fold range. This suggests that their exchange is mediated by a concerted transient unfolding reaction. The removal of allosterically important salt links at the distant alpha subunit N termini (des-Arg141 alpha hemoglobin) has little if any effect on the indicator NH at the beta C terminus. This demonstrates the restricted reach of the separate allosteric interactions in the T form as well as the localized nature of the H-exchange probe. Breakage of a salt link at the beta chain C terminus (His146 beta to Asp94 beta) by chemical modification (NES-Cys93 beta hemoglobin) speeds exchange of the indicator peptide NH in T-state hemoglobin by six-fold, which corresponds to an allosteric destabilization at the C-terminal segment of 1 kcal (pH 7.4, 0 degrees C), according to local unfolding theory. This is in quantitative agreement with energy values obtainable from other measurements. These NH exchange with an average halftime of five hours in deoxy hemoglobin and 15 seconds in oxy hemoglobin. According to the unfolding model for protein H-exchange, the 1200-fold increase in rate indicates a loss of 3.8 kcal in structural stabilization free energy at or near the C terminus of each beta chain in the T to R transition (pH 7.4, 0 degrees C, with 2,3-diphosphoglycerate). This result together with other available data places about 70% of hemoglobin's total allosterically significant structural energy change at the beta chain C termini.

Yeast iso-1-cytochrome c. A 2.8 A resolution three-dimensional structure determination.

A molecular replacement approach, augmented with the results of predictive modeling procedures, solvent accessibility studies, packing analyses and translational coefficient searches, has been used to elucidate the 2.8 A (1 A = 0.1 nm) resolution structure of yeast iso-1-cytochrome c. An examination of the polypeptide chain folding of this protein shows it to have unique conformations in three regions, upon comparison with the structures of other eukaryotic cytochromes c. These include: residues -5 to +1 at the N-terminal end of the polypeptide chain, which are in an extended conformation and project in large part off the surface of the protein; residues 19 to 26, which form a surface beta-loop on the His18 ligand side of the central heme group; and, the C-terminal end of the helical segment composed of residues 49 to 56, which serves to form a part of the heme pocket. Structural studies also show that the highly reactive sulfhydryl group of Cys102 is buried within a hydrophobic region in the monomer form of yeast iso-1-cytochrome c. Dimerization of yeast iso-1-cytochrome c through disulfide bond formation between two such residues would require a substantial conformational change in the C-terminal helix of this protein. Another unique structural feature, the trimethylated side-chain of Lys72, is located on the surface of yeast iso-1-cytochrome c near the solvent-exposed edge of the bound heme prosthetic group. On the basis of the results of these and other structural studies, an analysis of the spatial conservation of structural features in the heme pocket of eukaryotic cytochromes c has been conducted. It was found that the residues involved could be divided into three general classes. The current structural analyses and additional modeling studies have also been used to explain the altered functional properties observed for mutant yeast iso-1-cytochrome c proteins.

High-resolution three-dimensional structure of horse heart cytochrome c.

The 1.94 A resolution three-dimensional structure of oxidized horse heart cytochrome c has been elucidated and refined to a final R-factor of 0.17. This has allowed for a detailed assessment of the structural features of this protein, including the presence of secondary structure, hydrogen-bonding patterns and heme geometry. A comprehensive analysis of the structural differences between horse heart cytochrome c and those other eukaryotic cytochromes c for which high-resolution structures are available (yeast iso-1, tuna, rice) has also been completed. Significant conformational differences between these proteins occur in three regions and primarily involve residues 22 to 27, 41 to 43 and 56 to 57. The first of these variable regions is part of a surface beta-loop, whilst the latter two are located together adjacent to the heme group. This study also demonstrates that, in horse cytochrome c, the side-chain of Phe82 is positioned in a co-planar fashion next to the heme in a conformation comparable to that found in other cytochromes c. The positioning of this residue does not therefore appear to be oxidation-state-dependent. In total, five water molecules occupy conserved positions in the structures of horse heart, yeast iso-1, tuna and rice cytochromes c. Three of these are on the surface of the protein, serving to stabilize local polypeptide chain conformations. The remaining two are internally located. One of these mediates a charged interaction between the invariant residue Arg38 and a nearby heme propionate. The other is more centrally buried near the heme iron atom and is hydrogen bonded to the conserved residues Asn52, Tyr67 and Thr78. It is shown that this latter water molecule shifts in a consistent manner upon change in oxidation state if cytochrome c structures from various sources are compared. The conservation of this structural feature and its close proximity to the heme iron atom strongly implicate this internal water molecule as having a functional role in the mechanism of action of cytochrome c.

Energetic components of the allosteric machinery in hemoglobin measured by hydrogen exchange.

A hydrogen exchange (HX) functional labeling method was used to study allosterically active segments in human hemoglobin (Hb) at the alpha-chain N terminus and the beta-chain C terminus. Allosterically important interactions that contact these segments were removed one or more at a time by mutation (Hbs Cowtown, Bunbury, Barcelona, Kariya), proteolysis (desArg141alpha, desHis146beta), chemical modification (N-ethylsuccinimidyl-Cys93beta), and the withdrawal of extrinsic effectors (phosphate groups, chloride). The effects of each modification on HX rate at the local and the remote position were measured in the deoxy Hb T-state and translated into change in structural free energy at each position.The removal of individual salt links destabilizes local structure by 0.4 to 0.75 kcal/mol (pH 7.4, 0 degreesC, 0.35 M ionic strength) and often produces cross-subunit effects while hemoglobin remains in the T-state. In doubly modified hemoglobins, different changes that break the same links produce identical destabilization, changes that are structurally independent show energetic additivity, and changes that intersect show energetic overlap. For the overall T-state to R-state transition and for some but not all modifications within the T-state, the summed loss in stabilization free energy measured at the two chain termini matches the total loss in allosteric free energy measured by global methods. These observations illustrate the importance of evaluating the detailed energetics and the modes of energy transfer that define the allosteric machinery.

Crystallization and preliminary X-ray investigation of Escherichia coli porphobilinogen deaminase.

Porphobilinogen deaminase, the polymerase that catalyses the synthesis of preuroporphyrinogen, the linear tetrapyrrole precursor of uroporphyrinogen III, has been crystallized from sodium acetate buffer with polyethylene glycol 6000 as precipitant. The crystals are orthorhombic and the space group is P2(1)2(1)2, with unit cell dimensions a = 88.01 A, b = 75.86 A, c = 50.53 A and alpha = beta = gamma = 90 degrees, indicating a single molecule of 34 kDa in the asymmetric unit. The crystals grow to dimensions of 1 mm x 2 mm x 0.5 mm within two weeks in the dark and are stable in the X-ray beam for at least 40 hours. Diffraction data beyond 1.7 A resolution, observed with a synchrotron radiation source, indicate that a high resolution structure analysis is feasible.

The three-dimensional structures of mutants of porphobilinogen deaminase: toward an understanding of the structural basis of acute intermittent porphyria.

Mutations in the human gene for the enzyme porphobilinogen deaminase give rise to an inherited disease of heme biosynthesis, acute intermittent porphyria. Knowledge of the 3-dimensional structure of human porphobilinogen deaminase, based on the structure of the bacterial enzyme, allows correlation of structure with gene organization and leads to an understanding of the relationship between mutations in the gene, structural and functional changes of the enzyme, and the symptoms of the disease. Most mutations occur in exons 10 and 12, often changing amino acids in the active site. Several of these are shown to be involved in binding the primer or substrate; none modifies Asp 84, which is essential for catalytic activity.

Gln-41 is intermolecularly cross-linked to Lys-113 in F-actin by N-(4-azidobenzoyl)-putrescine.

The bifunctional reagent N-(4-azidobenzoyl)-putrescine was synthesized and covalently bound to rabbit skeletal muscle actin. The incorporation was mediated by guinea pig liver transglutaminase under conditions similar to those described by Takashi (1988, Biochemistry 27, 938-943); up to 0.5 M/M were incorporated into G-actin, whereas F-actin was refractory to incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln-41. The labeled G-actin was polymerized, and irradiation of the F-actin led to covalent intermolecular cross-linking. A cross-linked peptide complex was isolated from a tryptic digest of the cross-linked actin in which digestion was limited to arginine; sequence analysis as well as mass spectrometry indicated that the linked peptides contained residues 40-62 and residues 96-116, and that the actual cross-link was between Gln-41 and Lys-113. Thus the gamma-carboxyl group of Gln-41 must be within 10.7 A of the side chain (probably the amino group) of Lys-113 in an adjacent actin monomer. In the atomic model for F-actin proposed by Holmes et al. (1990, Nature 347, 44-49), the alpha-carbons of these residues in adjacent monomers along the two-start helices are sufficiently close to permit cross-linking of their side chains, and, pending atomic resolution of the side chains, the results presented here seem to support the proposed model.

Tight binding affinities determined from thermodynamic linkage to protons by titration calorimetry.

A general titration calorimetry method is described that can be used to determine the affinity of tight binding interactions with proteins. The method is based on the thermodynamic linkage between ligand binding and coupled protonation reactions. The protons linked to a given ligand-binding reaction are measured by titration calorimetry, and integration of the resulting data set yields the pH dependence of the binding affinity based on thermodynamic relationships developed elsewhere. When the pH dependence of the binding affinity is combined with the absolute affinity determined independently at a pH at which the affinity can be conveniently measured, the absolute binding affinity over the entire pH range is determined. The method is well suited for determining high-affinity binding interactions of protein antigens with antibodies, but is applicable to any macromolecular ligand-binding reaction that is coupled to protonation.

Aminophylline shortens thiopental sleep-time and enhances noradrenergic neurotransmission in rats.

We investigated the effect of aminophylline on thiopental sleep-times and monoamine neurotransmitter turnover rates in discrete brain areas. Aminophylline-treated rats had shorter thiopental sleep-times than saline-treated controls. Noradrenergic neurotransmission was greater following aminophylline treatment in thiopental-anesthetized rats in all brain areas while turnover in other monoaminergic pathways was unchanged. These data suggest that acute aminophylline treatment increases central noradrenergic neurotransmission which pharmacodynamically diminishes the hypnotic response to thiopental.

L-phenylisopropyladenosine (L-PIA) diminishes halothane anesthetic requirements and decreases noradrenergic neurotransmission in rats.

The effect of L-phenylisopropyladenosine (L-PIA), the A1 adenosine agonist, on the depth of anesthesia was investigated in halothane-anesthetized rats. L-PIA treatment reduced the minimum anesthetic concentration (MAC) of halothane that prevented 50% of animals from moving in response to a painful stimulus by 49%. MAC experiments performed with L-PIA given in conjunction with A1 adenosine receptor antagonists which either permeate the blood-brain barrier (8-phenyltheophylline [8-PT] or do not (8-sulphophenyltheophylline [8-So-PT]) indicate that central mechanisms are involved. Noradrenergic neurotransmission was diminished following L-PIA administration in halothane-anesthetized rats in all brain regions. These data suggest that acute L-PIA treatment decreases central noradrenergic neurotransmission and may represent the mechanism for the decrease in halothane dose to achieve an anesthetic endpoint anesthetic response to halothane.

Ewing's sarcoma: primary involvement of the zygoma undergoing resection and immediate reconstruction.

Because the occurrence of primary Ewing's sarcoma in the facial bones is unusual, it may pose diagnostic and therapeutic problems. Lack of clinical suspicion along with atypical radiographic features may lead to a delayed diagnosis. Furthermore, because of the limited number of cases, precise treatment guidelines are lacking. In our patient, whom we believe to be the first reported with primary Ewing's sarcoma originating in the zygoma, the tumor was successfully managed surgically. A combination of craniofacial and microsurgical principles made surgical resection and immediate reconstruction possible in an area not generally thought to be amenable to surgery; moreover, we thus avoided the potential deleterious effects of radiation in the facial region in a growing child.

Trajectory of white matter hyperintensity burden preceding mild cognitive impairment.

OBJECTIVE: To determine the time of acceleration in white matter hyperintensity (WMH) burden, a common indicator of cerebrovascular pathology, in relation to conversion to mild cognitive impairment (MCI) in the elderly. METHODS: A total of 181 cognitively intact elderly volunteers from the longitudinal, prospective, Oregon Brain Aging Study underwent yearly evaluations, including brain MRI, and cognitive testing. MRIs were analyzed for imaging markers of neurodegeneration: WMH and ventricular CSF (vCSF) volumes. The time before MCI, when the changes in WMH and vCSF burden accelerate, was assessed using a mixed-effects model with a change point for subjects who developed MCI during follow-up. RESULTS: During a follow-up duration of up to 19.6 years, 134 subjects converted to MCI. Acceleration in %WMH volume increase occurred 10.6 years before MCI onset. On average, the annual rate of change in %WMH increased an additional 3.3% after the change point. Acceleration in %vCSF volume increase occurred 3.7 years before the onset of MCI. Out of 63 subjects who converted to MCI and had autopsy, only 28.5% had Alzheimer disease (AD) as the sole etiology of their dementia, while almost just as many (24%) had both AD and significant ischemic cerebrovascular disease present. CONCLUSIONS: Acceleration in WMH burden, a common indicator of cerebrovascular disease in the elderly, is a pathologic change that emerges early in the presymptomatic phase leading to MCI. Longitudinal changes in WMH may thus be useful in determining those at risk for cognitive impairment and for planning strategies for introducing disease-modifying therapies prior to dementia onset.

Functional evaluation of the shoulder following latissimus dorsi muscle transfer.

The dynamic muscle function of the shoulder in 26 patients (10 males, 16 females) who underwent a pedicled or free vascularized latissimus dorsi muscle transfer between 1985 and 1991 (mean follow-up, 4.4 yr) was studied. Instrumented muscle testing was performed on the Kinetic Communicator machine (Kin Com) and the Baltimore Therapeutic Equipment (BTE) work simulator. The female unilateral pedicle group (n = 13) showed a significant difference between operated and nonoperated shoulders for both peak torque (power) and work (endurance) measurements of shoulder adduction and extension on the Kin Com (mean ratios operated/nonoperated shoulders, 55% to 69%). They also showed significant differences for work performance on three of four BTE tests (mean ratios, 77% to 84%). The male free vascularized group (n = 10) similarly showed a significant deficit of both peak torque and work for shoulder extension and adduction on the Kin Com (mean ratios, 74% to 84%); however, they showed no deficit on the BTE tests. In conclusion, dynamic muscle tests demonstrate a deficit of muscle power and endurance of shoulder extension and adduction following latissimus dorsi muscle transfer.