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Biomedical subjects

C Woodward

Publications and source records attributed to C Woodward.

At least 37 records · Page 2Linked to original sources
PubMed1998

Feasibility of orthopaedic teleconsulting in a geriatric rehabilitation service.

Fifteen elderly patients participated in a teleconsultation with an orthopaedic surgeon, which was then followed by a conventional, face-to-face consultation. The comparison between the surgeon's ratings for both types of consultation suggested that the telemedicine consultation was satisfactory in terms of the quality of image and sound, the clinical examination and general simplicity. The telemedicine consultations did not generate a need for any additional clinical investigations, although in two cases a face-to-face consultation was necessary to clarify clinical signs (shortening of a limb and scar tissue). The surgeon's rating of his decision level was superior in the face-to-face situation in four cases, and for 11 patients it was equal. Similarly, the surgeon's level of confidence in decision making was superior in the conventional situation for five patients and equal for 10 patients. Patient attitudes towards teleconsulting were favourable. There was a high level of patient satisfaction. Teleconsulting between orthopaedic surgeons and elderly patients therefore appears to be possible, provided that certain technical, clinical and psychological considerations are addressed.

Aged↗
PubMed1997

A pulsed-field gradient NMR study of bovine pancreatic trypsin inhibitor self-association.

Previous studies have produced conflicting interpretations regarding the aggregation state of BPTI in solution. Here, pulsed-field gradient NMR self-association measurements have been performed with BPTI under a variety of temperature, pH, salt, urea conditions, and protein concentrations. Relative to the standard proteins, lysozyme, ribonuclease, and ubiquitin, diffusion constants indicate that BPTI dimerizes at concentrations above about 3 mg/mL and below 280 K. At higher temperatures, a marked self-association is observed above 10 mg/mL. The apparent lack of significant effects from variations in pH and NaCl concentration suggests minimal contribution to the aggregation process from charge-charge interactions. In contrast, in nondenaturing concentrations of urea (2 M), BPTI behaves as a monomer, suggesting that hydrophobic and polar residues modulate BPTI association. The BPTI surface shows that while one side is highly charged, the opposite side, composed mostly of hydrophobic and some hydrophilic residues, is feasible as an interface for BPTI self-association.

Animals↗
PubMed1997

Local fluctuations and global unfolding of partially folded BPTI detected by NMR.

The protein [14-38]Abu is a chemically synthesized variant of bovine pancreatic trypsin inhibitor (BPTI) with the 14-38 disulfide bond intact and cysteines 5, 30, 51, and 55 replaced by alpha-amino-n-butyric acid (Abu). At 1-6 degrees C and pH 4.5-6.5, [14-38]Abu is partially folded with a native-like core [1]. Heteronuclear NMR spectra contain two, and in a few cases three or four, exchange cross peaks for each 15N-bound 1H, reporting the presence of two or more conformations that interconvert on a time scale of > or = milliseconds. Thermodynamic analysis of 15N-1H exchange peak volumes as a function of temperature in the range 1-35 degrees C indicates that partially folded [14-38]Abu undergoes local segmental motions as well as cooperative global unfolding. The relative abundance of more folded versus disordered conformations changes throughout the molecule, indicating that various regions of the partially folded protein are disordered to different extents prior to onset of thermal denaturation. This system is unique in providing a measure of the populations of interconverting partially folded conformations, as well as a microscopic view of cooperative folding of a fluctuating ensemble. Although global thermal denaturation is cooperative, significant deviation from simple two-state behavior is reflected in several parameters, including the difference in Tm for thermal unfolding measured by NMR versus circular dichroism.

Aprotinin↗
PubMed1997

Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor.

Pulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R]Abu, at pH 4.5 and pH 2.5, and unfolded [14-38]Abu, at pH 2.5. These were prepared by chemical synthesis. [R]Abu is a model for reduced BPTI; all cysteine residues are replaced by alpha-amino-n-butyric acid (Abu). [14-38]Abu retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of D degree -the diffusion coefficient extrapolated to infinite dilution-is determined. From D degree, a value of the hydrodynamic radius. Rh, is computed from the Stokes-Einstein relationship. At pH 4.5, [R]Abu has an Rh value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental Rh value is the same as for a random coil. In view of the changes in NMR detected structure of [R]Abu at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non-random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:13974-13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non-native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14-38]Abu was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14-38]Abu, relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under "molten globule" conditions.

Aprotinin↗
PubMed1997

[Value and limitations of pulmonary scintigraphy/venous duplex Doppler echography in the management of pulmonary embolism].

PURPOSE: To assess the accuracy of diagnostic strategy of pulmonary embolism (PE) based on clinical examination, lung scan and venous duplex US findings. METHODS: 1,819 patients have been included in a prospective study (mean age: 66, range: 6-102, F 54% H 46%) over a 13 month period. RESULTS: To decide the opportunity of anticoagulant therapy, lung scan alone is decisive in 30.6% of the cases. When taking into account clinical examination, lung scan and venous duplex US findings in a combined diagnostic strategy, a therapeutic decision can be made for 74.2% of the patients. The decisive characteristics of this strategy were influenced by two factor: age (therapeutic decision can be reached for 83% of the patients aged 30 to 50 vs 65% when they are over 85, p < 0.01); history of heart or pulmonary disease (therapeutic decision reached in 62% of the cases with history vs 78% without, p < 0.01). CONCLUSION: Pulmonary angiography seems theoretically necessary in less than 26% of the patients with suspected PE when they have undergone lung scan and venous duplex US. In this case, and when these strategies are not very decisive, it would be important to assess the diagnostic value of spiral CT scanning.

Adolescent↗
PubMed1997

Diagnosis of photosensitivity to flupenthixol by photoprick testing.

We report the case of a 38-year-old patient with an erythematous eruption, initially confined to photo-exposed areas and then becoming more generalized, which was attributable to flupenthixol, a thioxanthene derivative used for its neuroleptic properties. The compound has the same polycyclic aromatic hydrocarbons as phenothiazines, but without any cross-reaction in our patient. A photoallergic cause for the eruption was verified on clinical, histological and photobiologic grounds. Photopatch tests were negative, but photoprick testing was positive after UV irradiation of the test site. Photopatch test methods using better skin penetration have been already reported for testing drug photosensitivity. Photoprick testing combines better penetration with greater ease of use.

Adult↗
PubMed1997

Isolation and characterization of rcsB mutations that affect colanic acid capsule synthesis in Escherichia coli K-12.

Regulation of colanic acid polysaccharide capsule synthesis in Escherichia coli requires the proteins RcsC and RcsB, in addition to several other proteins. By sequence similarity, these two proteins appear to be members of the two-component sensor-effector regulatory family found in bacteria. The present study characterizes the functional domains of RcsB. We have isolated mutations in rcsB that are able to suppress an rcsC "up" mutation (i.e., leading to increase in cps transcription) that normally results in constitutive expression of the capsule. In addition, constitutive capsule mutations in rcsB have been isolated. From the characterization of the mutants and by analogy to the three-dimensional structure of CheY, we have begun to define different domains of RcsB and to assign functions to them. A few of the constitutive capsule mutations were localized in an acidic pocket that has been proposed to play a crucial role in phosphorylation of RcsB. As seen in other two-component systems, an aspartate-to-glutamate substitution at the presumed site of phosphorylation of RcsB resulted in constitutive capsule expression. Lastly, several of our rcsB mutants were found to be allele specific (rcsC137 specific) for rcsC, suggesting a physical as well as functional interaction between RcsC and RcsB proteins.

Amino Acid Sequence↗
PubMed1996

Unfolded BPTI variants with a single disulfide bond have diminished non-native structure distant from the crosslink.

BACKGROUND: NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions favored in initial stages of folding, and in early intermediates formed during folding. We have characterized non-random structures favored in unfolded, reduced BPTI [1], and in partially folded BPTI [2]. Here, we report NMR-detected structure of two analogs of unfolded BPTI with one native 14-38 disulfide bond. RESULTS: Analogs Y21A[14-38]Abu and Y23A[14-38]Abu, obtained by chemical synthesis of [14-38]Abu with Y21 or Y23 replaced by alanine, are models for unfolded BPTI with 14-38 the only disulfide. Compared to unfolded BPTI with all three disulfides broken, the unfolded 14-38 BPTI analogs have numerous differences, including loss of non-native, turn-like conformations for beta 2 residues, diminished non-native aromatic-aliphatic NOEs, and increased intermediate chemical exchange of residues that have native-like conformations in partially folded BPTI. Although the Y21A and Y23A analogs have similar CD and NMR properties, specific differences in NOE patterns and in exchange broadening are observed. CONCLUSIONS: Changes in unfolded BPTI associated with formation of the 14-38 disulfide bond are consistent with less non-native structure, and more native-like structure, in residues composing the stable core of antiparallel beta-sheet in partially folded BPTI. Specific differences between Y21A[14-38]Abu and Y23A[14-38]Abu indicate that replacement of Y23 results in less ordered structure than replacement of Y21.

Amino Acid Sequence↗
PubMed1995

Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor.

Two-dimensional 1H NMR spectra of an analog of reduced BPTI at pH 4.5, 1 degrees C, have been assigned. Spectra indicate considerable conformational averaging, as expected for a flexible, unfolded protein. The presence of extensive nonrandom structure is detected by the presence of NHi-NHi + 1 and aromatic-aliphatic NOEs. Sequential amide-amide NOEs indicate that turn-like conformations are significantly populated at 18 pairs of residues along the chain. Many of these are located in a turn, loop, or helix in native BPTI, but six are observed for contiguous pairs in the segment composed of residues 29-35, which in native BPTI constitute a strand of extended sheet. A novel finding for unfolded proteins is our observation of NOEs implying non-native hydrophobic interactions. Multiple aromatic-aliphatic NOEs are observed for pairs of residues that are within 1-3 residues of each other. Most are non-native and involve residues in both strands of the central antiparallel strand-turn-strand of native BPTI comprised of residues 18-35. All NOEs reported for oligopeptides spanning the BPTI sequence [Kemmink, J., & Creighton, T. (1993) J. Mol. Biol. 234, 861-878] are observed in reduced BPTI, but many others are present as well. Similar spectra are obtained for naturally occurring BPTI reduced by dithiothreitol, BPTI with cysteines replaced by alpha-amino-n-butyric acid, and BPTI mutant F45A reduced by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Dynamic structure of a highly ordered beta-sheet molten globule: multiple conformations with a stable core.

The structure of [14-38]Abu, a variant of bovine pancreatic trypsin inhibitor (BPTI) with only the 14-38 disulfide bridge intact, has been analyzed by two-dimensional 1H and 1H-15N NMR. Except for the 18-24, 29-35 antiparallel beta-sheet, residues in all regions of the molecule give two exchange cross peaks for each 1H; for one residue, Gly 37, three exchange cross peaks are observed. The presence of exchange cross peaks indicates that the residues sample conformations that interconvert on a time scale of milliseconds or longer. Over 90% of the NMR spectra have been assigned, including backbone and side chain atoms and their exchange cross peaks. Analyses of chemical shifts, chemical exchange, hydrogen isotope exchange, and NOEs indicate that [14-38]Abu at pH 4.5 and 1 degree C is an ensemble of interconverting conformations, in all of which the 18-24, 29-35 antiparallel beta-sheet is native-like and intact. Outside the antiparallel beta-sheet, residues undergo local order/disorder transitions. The stable structure of [14-38]Abu is not in the vicinity of the 14-38 disulfide bond but rather is in the slow-exchange core. NOE analysis indicates that the main tertiary interactions involve hydrophobic contacts with the rings of Tyr 21, Tyr 23, and Tyr 35. As a model for early folding intermediates, the structure of [14-38]Abu suggests that BPTI folding is initiated by stabilization of a turn existing in the unfolded protein and involves both local and nonlocal hydrophobic interactions.

Amino Acid Sequence↗
PubMed1995

Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa > 9.

Apparent pKa values of active site residues Asp26, Cys32, and Cys35 in reduced thioredoxin have been characterized. Both wild-type thioredoxin and mutant D26A thioredoxin were selectively 13C-enriched on cysteine beta-carbons. In both proteins, the variation with pH of 1HB1, 1HB2, and 13CB NMR chemical shifts has been measured. In wild-type reduced thioredoxin, for both cysteines, the pH versus chemical shift plots of HB1 protons can be fit to one titration with pKa values of 7.0-7.1. In contrast, the HB2 protons and beta-carbons give pH--chemical shift plots that clearly reflect more than one titration; fits to the data give apparent pKa values of 7.0-7.3 and 9.5 for HB2 protons and 7.5-7.9 and 9.2-10.2 for CB carbons. In reduced D26A, all three probe chemical shifts have a pH dependence that is fit by one titration with pKa of 7.4-7.9. The absence of a titration with pKa > 9 in D26A, taken together with cysteine thiol pKa values of 7.1 and 7.9 determined by Raman spectroscopy [Li et al. (1993) Biochemistry 32, 5800-5808], indicates that the pKa > 9 in reduced thioredoxin is that of Asp26. This is highly significant in view of the previous observation that, in oxidized thioredoxin, Asp26 pKa is 7.5 [Langsetmo et al. (1991) Biochemistry 30, 7603-7609]. The very high pKa values of these carboxyls is consistent with their local environment in the three-dimensional structure; the Asp26 side chain in oxidized thioredoxin is almost but not completely buried, and in reduced thioredoxin it may be even more buried.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗