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Biomedical subjects

J Clarke

Publications and source records attributed to J Clarke.

At least 163 records · Page 9Linked to original sources
PubMed1996

Substitution of specific amino acids in insulin-like growth factor (IGF) binding protein 5 alters heparin binding and its change in affinity for IGF-I response to heparin.

Heparin binding to insulin-like growth factor (IGF)-binding protein 5 (IGFBP-5) leads to a 17-fold decrease in its affinity for IGF-I, and a region that contains several basic amino acids (Arg201-Arg218) may be involved in this affinity shift. In the present study, mutagenesis was used to analyze the effect of substitutions for basic amino acids in the Arg201-Arg218 region of IGFBP-5 on heparin-binding and the heparin-induced affinity shift. Nine mutant forms were prepared. Their association constants (Ka) for IGF-I were similar to native IGFBP-5. When 10 microg/ml of heparin was added, the Ka of native IGFBP-5 decreased 17-fold, and the Ka of the K134A/R136A mutant decreased 16-fold. In contrast, substitutions for specific basic amino acids in the Arg2O1-Arg218 region decrease the affinity shift to 1.1-3.2-fold. Lys 211 was especially important. When a mutant containing that single substitution was tested, heparin caused only a 2.5-fold reduction in IGF-I affinity. Affinity cross-linking studies showed that heparin was equipotent in inhibiting the formation of 125I-IGF-I-K134A/Rl36A mutant complexes compared to native IGFBP-5. In contrast, heparin had minimal effects on the formation of complexes between 125I-IGF-I and the other mutants. The heparin-binding activity of each mutant was determined. Four mutants, R201A/K202N, K202A/K206A/R207A, R201A/K202N/K206N/K208N, and K211N/R214A/K217A/R218A, had reduced heparin binding compared to native IGFBP-5. The other five mutants, including the K21IN mutant, showed no change in heparin binding. The four mutants with reduced heparin binding could be dissociated from heparin-Sepharose with much lower NaCl concentrations, indicating that they had reduced affinity. These findings suggest that Arg201 Lys202, LysS206, and Arg214 are important for heparin binding. In contrast, LyS211 is not important for the binding of IGFBP-5 to heparin, but substitution for it reduced the heparin-induced affinity shift.

Affinity Labels↗
PubMed1996

An evaluation of the use of hydrogen exchange at equilibrium to probe intermediates on the protein folding pathway.

BACKGROUND: Methods have been developed recently for probing local fluctuations of protein structure using H/2H-exchange of amide protons at equilibrium. It has been suggested that equilibrium exchange methods can identify the order of events in folding pathways and detect folding cores. We have applied the procedure of measuring the effects of denaturant on the H/2H-exchange of amide protons of barnase, the folding pathway of which is well established. RESULTS: The addition of relatively low concentrations of denaturant causes the mechanism of exchange of amide protons of barnase to change from EX2 to EX1 for the residues that require global unfolding for exchange to occur. This change of mechanism, which would have been missed by some of the standard tests, causes artefacts that could be easily misinterpreted. We also present the thermodynamic argument that measurements at equilibrium cannot give the order of events in folding. CONCLUSIONS: Measurement of H/2H-exchange of amide protons at equilibrium, when applied correctly, is an excellent method for analyzing the equilibrium distribution of unfolded and partly folded states. It cannot, in theory and in practice, be used for determining protein folding pathways by itself.

Amides↗
PubMed1996

Measuring outcomes: one month survival after acute myocardial infarction in Scotland.

OBJECTIVE: To examine 30 day survival after acute myocardial infarction as an outcome indicator, and explore the effects of adjusting for available prognostic factors such as age, sex, co-morbidity, deprivation, and deaths outside hospital. DESIGN: Cohort study. SETTING: The Scottish Record Linkage System was analysed. This national data-base links inpatient data to death certificate information for a population of 5.1 million. SUBJECTS: All 40,371 admissions to hospital with a principal diagnosis of acute myocardial infarction, plus all 18,452 deaths outside hospital with a principal cause of death registered as acute myocardial infarction (ICD9 code 410) during 1988-1991. MAIN OUTCOME MEASURES: The outcome event was death from any cause, within hospital or elsewhere, within 30 days of admission. RESULTS: During 1988-1991, 30 day survival after acute myocardial infarction was 77% in 40,371 hospital admissions, but only 53% when 18,452 acute myocardial infarction deaths in the community were included (a population-based outcome indicator with many advantages). Using logistic regression at an individual patient level, the odds of dying within 30 days effectively doubled for each decade of age (odds ratio compared with patients aged under 55: 2.3 aged 55-64, 4.4 aged 65-74, 8.2 aged 75-84, 12.0 aged 85 plus); were marginally higher in females than in males (odds ratio 1.07); were almost doubled in patients with a history of previous infarction, coronary heart disease, or other heart disease, and were also significantly increased in patients with circulatory disease, respiratory disease, neoplasm, or diabetes. Socioeconomic deprivation had no significant effect. Marked variations in survival between different hospitals and health board areas persisted, even after adjusting for the above prognostic factors. CONCLUSION: One month survival after acute myocardial infarction could be a useful means of measuring outcome of hospital care. There was important geographical variation in one month survival. These differences could be accounted for by variations in referral, admission, diagnosis, definition, and coding. These variables merit further research and local clinical audit before one month survival after acute myocardial infarction can be reliably used for detecting differences in quality of care. In addition, it would be essential to take account of infarct severity.

Age Factors↗
PubMed1996

Late effects of retinoic acid on neural crest and aspects of rhombomere.

We exposed st.10 chicks to retinoic acid (RA), both globally, and locally to individual rhombomeres, to look at its role in specification of various aspects of hindbrain derived morphology. Previous studies have looked at RA exposure at earlier stages, during axial specification. Stage 10 is the time of morphological segmentation of the hindbrain and is just prior to neural crest migration. Rhombomere 4 localised RA injections result in specific alterations of pathways some crest cells that normally migrate to sites of differentiation of neurogenic derivatives. The r4 crest cells that give rise to mesenchymal derivatives are unaffected. In addition, r4 gene expression is also partially altered by RA; within 6 hours of r4 exposure to RA, ectopic expression of Krox-20 is seen in r4 and Hoxb-1 expression is lost while Hoxa-2 expression continues normally. When we examined these RA-treated animals later in development, they showed an anterior displacement of the facial ganglion in addition to a mis-direction of the extensions of its distal axons and a dramatic decrease in the number of contralateral vestibuloacoustic neurons normally seen in r4. Only this r4-specific neuronal type is affected in r4; the motor neuron projections seem normal in experimental animals. The specificity of this result, combined with the loss of Hoxb-1 expression in r4 and the work by Krumlauf and co-workers showing gain of contralateral neurons co-localised with ectopic Hoxb-1 expression, indicates a role for Hoxb-1 and RA in the specification of this cell type in normal development. These results suggest that RA, at st.10, is able to affect some aspects of segment identity while leaving others unchanged.

Animals↗
PubMed1996

MRI in the evaluation of late bone marrow changes following bone marrow transplantation.

Measurements of MR spin-lattice (T1), and spin-spin (T2) relaxation times in lumbar vertebrae have been performed in a pilot study on six adult patients, treated for acute myeloid leukaemia (AML). All patients were treated with initial chemotherapy and then proceeded to bone marrow transplantation (BMT), conditioned with Melphalan and total body irradiation (TBI). MR measurements were made between 21 and 89 months after TBI. The relaxation times in the six patients were compared with those in six healthy age-matched volunteers to establish whether there were differences between the two groups. Average T1 values in the vertebrae of the treated patients are significantly shorter (p < 0.01) than in the healthy volunteers. This is consistent with the observation of a relatively hyperintense vertebral bone marrow in the T1 weighted images and is likely to be a consequence of treatment induced fatty replacement of marrow. Shorter T1 values tend to be distributed within the centre of the lumbar vertebrae compatible with observations, made by others, which suggest that the peripheral zone of the vertebral body has been repopulated with bone marrow cells whereas the central zone, around the basivertebral vein, is predominantly fat. Histogram displays of vertebral body relaxation time distributions (T1, T2) for both patients and healthy age-matched volunteers are similar in that both patients and volunteers give histograms that are only slightly skewed. This similarity is probably a reflection of the fact that the patients have been in remission for over a year and have generally healthy bone marrow.

Acute Disease↗
PubMed1996

Linking anatomy and physiology in modeling respiratory mechanics.

We present an integrated 3D virtual environment for the quantitative modeling of the anatomy and the physiology of the pulmonary system. Our approach formally integrates 3D deformable object modeling with conventional models of respiratory mechanics. We demonstrate quantitatively, aspects of the behavior of the respiratory system qualitatively known to clinicians, such as normal quiet breathing and an open sucking chest wound. Our methodology is general and can be used to model both the anatomy and the physiology at many levels of detail. Another important aspect of our approach is that based on our previously developed computer vision techniques we can make such a simulation patient specific. The usefulness of such a system is manifold. Medical education, surgical planning, disease diagnosis are some of the many areas such a system can be applied.

Computer Simulation↗
PubMed1995

Disulfide mutants of barnase. I: Changes in stability and structure assessed by biophysical methods and X-ray crystallography.

In this series of papers, we examine the effects of introducing disulfide bonds on the properties, structure and thermodynamics of a small globular protein, barnase. Three mutants have been made, in each of which a single crosslink confers different properties. Two of the disulfide bonds, between residues 43 and 80 (43-80) and between residues 85 and 102 (85-102), stabilise the protein, relative to both wild-type and the corresponding (reduced) dithiol forms: 85-102 is more stable than predicted from the entropic destabilisation of the unfolded state; 43-80 is less stable than predicted. The third disulfide bond, between residues 70 and 92 (70-92) destabilises the protein relative to both wild-type and the corresponding dithiol form, implying significant disruption of the folded protein on formation of the disulfide bond. Crystal structures of the three mutant proteins have been solved. All three proteins have essentially the same fold as wild-type, but with left-handed disulfide bonds, which have dihedral geometries that have not been observed in naturally occurring disulfides. In the very stable mutant 85-102, there is no significant difference between the mutant and wild-type structures: these data do not explain the large stability of this protein. The disulfide bond at 43-80 induces small structural rearrangements close to the site of the disulfide bond, associated with some local disorder: the crosslink appears to decrease the stability of the native form of the protein. The destabilising disulfide bond at 70-92 induces considerable structural change, with displacement of a loop and consequent disruption of a stabilising salt-bridge. Our studies do not support the view that the conformation of the disulfide bond is crucial in determining the stability of the mutant proteins.

Bacillus↗
PubMed1995

Disulfide mutants of barnase. II: Changes in structure and local stability identified by hydrogen exchange.

The hydrogen exchange behaviour of two stabilised disulfide mutants of barnase has been examined using NMR H/2H exchange measurements. The choice of experimental conditions is crucial in experiments to study the effects of mutations on local and global stability and dynamic behaviour of proteins. If exchange conditions allow both local and global unfolding events to be examined, then a comparison of three proteins (two mutants and wild-type) allows the effect of a mutation on the folded state to be ascribed to specific increases or decreases in local stability. This method was used to examine the effect of an introduced crosslink on the folded state of two different disulfide mutants of barnase, and the results are related to structural studies. It is found that disulfide bonds can stabilise or destabilise local structures as well as having an effect upon global stability. The effects of the mutations on exchange rate constants of protons are compared with the effects on the structure upon the introduction of the disulfide bonds. In the protein with a disulfide bond between residues 43 and 80, some local exchange rate constants are higher, reflecting local destabilisation at the site of the disulfide bond, associated with small structural rearrangements. In the protein with a disulfide bond between residues 85 and 102, amide protons of the adjacent loop are protected to a considerable extent. This is not associated with a structural rearrangement yet indicates that this disulfide bond has an effect on the stability of this loop.

Bacillus↗
PubMed1995

Relationship between equilibrium amide proton exchange behavior and the folding pathway of barnase.

We describe a three-part strategy for analyzing the relationship between equilibrium amide proton exchange behavior of barnase and its folding/unfolding pathway. First, the effects of mutation on stability and kinetics are compared to reveal which residues exchange by local breathing, which by local unfolding, and which by a mixture of the two mechanisms. Second, is to detect any change of mechanism between EX2 and EX1 from the pH dependence of exchange and its relationship to structure and kinetics. The third is to determine from which state exchange takes place for residues that nominally exchange by a global process: the fully unfolded state or the folding intermediate. Experiments were performed at values of pH and temperature around physiological and close to conditions under which the folding pathway of barnase has been studied in detail. A set of residues was found for which the rate constants for exchange change on mutation by exactly the same factor as does the equilibrium constant for unfolding. Further, the protection factor against exchange for these residues in wild-type barnase is very similar to the equilibrium constant for overall folding measured by differential scanning calorimetry and extrapolated to the identical reaction conditions. These residues clearly exchange by a global unfolding mechanism, and the protection factors are consistent with the denatured state of barnase being largely as unprotected as model peptides. The rate constants for exchange of a second set of residues are unaffected by distant mutations, and so these exchange by local breathing. The logarithms of the rate constants (log kex) increase linearly with pH for the locally exchanging residues, consistent with the kinetics of the EX2 mechanism at these values of pH. The pH dependence for the globally exchanging residues, however, indicates a switch away from EX2 between pH 6.7 and 7.9 at 37 degrees C. The state from which "global" exchange occurs was probed also by using mutants in which the folded state of each is destabilized by the same amount by mutation relative to the unfolded state but the destabilization of the folding intermediate varies considerably. Under EX2 conditions, the changes in kex for all these residues follow the overall destabilization, confirming that exchange occurs from the fully unfolded state, not from the folding intermediate. The common characteristic of the residues that exchange by global unfolding is that they are all buried within the protein.

Amides↗
PubMed1995

Pulmonary hypertension in infants with chronic lung disease: non-invasive evaluation and short term effect of oxygen treatment.

A total of 45 examinations on 30 premature infants with chronic lung disease (CLD) of prematurity were made using Doppler echocardiography. Pulmonary systolic time intervals and tricuspid regurgitant velocity were measured to assess the prevalence of pulmonary hypertension and short term responsiveness of the pulmonary circulation to oxygen. Twelve preterm infants matched for gestational age, served as controls. Tricuspid regurgitation (TR) was detected in 14 of the patients. Eleven of those with TR had pulmonary hypertension, of whom eight responded to oxygen treatment. Of the remaining three patients with 'fixed' pulmonary hypertension, one subsequently died. The detection of TR was the basis of the preferred method for measuring pulmonary artery pressure (Ppa) non-invasively, but the degree of correlation between the Ppa estimated from TR and pulmonary systolic time intervals was high (r = -0.84, p = 0.001). In the absence of TR, systolic time intervals are an effective way to monitor pulmonary artery pressure in infants with CLD. Without these measurements, it would have been impossible to predict which subjects had pulmonary hypertension, and which might respond to oxygen treatment.

Blood Pressure↗