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PubMed1998

Radiolabelled mixed leukocytes and pure granulocytes with stabilized 99Tcm-exametazime.

Although the methylene blue stabilizer extends the shelf life of 99Tcm-exametazime to 4-6 h after reconstitution, the dark blue appearance of the mixture of stabilized 99Tcm-exametazime and blood components makes it impossible to separate out the leukocyte button. The aim of this study was to assess the feasibility of using stabilized 99Tcm-exametazime to radiolabel mixed leukocytes separated by Volex sedimentation with hypotonic lysis (VL) and pure granulocytes isolated by a single-density Ficoll-Hypaque gradient with hypotonic lysis (FL). Isolated cells from 40-ml and 80-ml donor blood samples were mixed with 0.5 ml stabilized 99Tcm-exametazime (approximately 925 MBq 99Tcm and 62.5 micrograms exametazime) and incubated at room temperature for 15 min. After incubation, two dilution steps with 3 ml and 9 ml of 12.6% ACD/NS (anticoagulant citrate dextrose, solution A, USP, mixed with 0.9% NaCl, v/v) were conducted to dilute the dark blue mixture and to remove any unbound 99Tcm activity. With the addition of 9 ml of 12.6% ACD/NS solution to the 1-ml bottom portion from the first dilution, the supernatant of the centrifuged preparation was clear enough to be withdrawn. The overall labelling efficiency (LE) of labelled leukocytes and granulocytes was 87.1 +/- 4.9% and 87.7 +/- 6.2%, respectively (n = 12 each). Overall, radiolabelled cells (n = 12) from the 80-ml blood samples (LE = 90.3 +/- 2.8%) had an approximately 6% higher labelling efficiency than from the 40-ml blood samples (LE = 84.5 +/- 6.0%) and also had a slightly better in vitro stability compared to the 40-ml samples. The in vitro stability studies showed that only approximately 2% (n = 48) 99Tcm activity was eluted each hour from the radiolabelled leukocytes or granulocytes for the 40-ml or 80-ml blood samples during the 6-h evaluation period. Cell viability of all labelled leukocyte samples was confirmed by the trypan blue staining technique. In conclusion, mixed leukocytes separated by the VL method and pure granulocytes isolated by the FL method can be effectively labelled with stabilized 99Tcm-exametazime with the use of the 'double dilution' technique.

Cell Survival↗
PubMed1994

Visual stabilization of posture in the elderly: fallers vs. nonfallers.

BACKGROUND: Postural instability is one of the important contributors to falling in observers aged 65 years and older. In this study we examine the role of vision in the relation between postural stability and falling, as well as in the relation between postural stability and the fear of falling. METHODS: Community-dwelling adults 65 years and older were administered a questionnaire about their history of falls and fear of falling (N = 185). Postural sway was measured in the same subjects with eyes open and eyes closed. Visual function was assessed by measures of visual acuity and contrast sensitivity. RESULTS: The 29 subjects who reported falling in the last year showed less of a visual contribution to posture stabilization than those who reported no falls. Controlling for age and gender, there is a 2.13-fold increase in the likelihood of reporting falling for a 0.1 decrement in the visual stabilization index when it is measured within the context of reliable somatosensory feedback. Those who reported a fear of falling and those who reported no fear showed similar visual stabilization. Contrast sensitivity was significantly associated with visual stabilization when it was measured within the context of reliable somatosensory feedback. CONCLUSIONS: The visual contribution to postural stabilization is significantly greater in nonfallers compared to fallers, and it is significantly associated with contrast sensitivity.

Accidental Falls↗
PubMed2000

Stability analysis of two linear accommodation and convergence models.

PURPOSE: To perform time domain stability analysis on two widely accepted linear models of accommodation and convergence. METHODS: For each model, the governing differential equations were used to compute the system matrix. The system matrices were used to calculate the respective trace and determinant from which eigen-vectors and eigenvalues were quantified. These characteristic numbers fully identified and classified the fixed points of each model and, thus, their stability. RESULTS: Controller gains, time constants, and accommodation and convergence cross-links determined model stability. Accommodation and convergence cross-links have the greatest influence on stability. A model-specific transition between stabilities was identified as the product of these cross-links, AC.CA(CRITICAL). For each model, three types of fixed points are described and displayed graphically: stable node, line of nonisolated fixed points, and saddle. CONCLUSION: We demonstrated the stability analysis of a two-dimensional linear system, working only in the time domain. The benefit of time-domain stability analysis is that it can be extended to nonlinear systems when frequency domain analysis techniques fail. Given that we live in a fundamentally nonlinear world and that the use and application of computational models is extensive, this is a valuable and important tool.

Accommodation, Ocular↗
PubMed1997

Stabilizing function of trunk flexor-extensor muscles around a neutral spine posture.

STUDY DESIGN: This study examined the coactivation of trunk flexor and extensor muscles in healthy individuals. The experimental electromyographic data and the theoretical calculations were analyzed in the context of mechanical stability of the lumbar spine. OBJECTIVES: To test a set of hypotheses pertaining to healthy individuals: 1) that the trunk flexor-extensor muscle coactivation is present around a neutral spine posture, 2) that the coactivation is increased when the subject carries a load; and 3) that the coactivation provides the needed mechanical stability to the lumbar spine. SUMMARY OF BACKGROUND DATA: Theoretically, antagonistic trunk muscle coactivation is necessary to provide mechanical stability to the human lumbar spine around its neutral posture. No experimental evidence exists, however, to support this hypothesis. METHODS: Ten individuals executed slow trunk flexion-extension tasks, while six muscles on the right side were monitored with surface electromyography: external oblique, internal oblique, rectus abdominis, multifidus, lumbar erector spinae, and thoracic erector spinae. Simple, but realistic, calculations of spine stability also were performed and compared with experimental results. RESULTS: Average antagonistic flexor-extensor muscle coactivation levels around the neutral spine posture as detected with electromyography were 1.7 +/- 0.8% of maximum voluntary contraction for no external load trials and 2.9 +/- 1.4% of maximum voluntary contraction for the trials with added 32-kg mass to the torso. The inverted pendulum model based on static moment equilibrium criteria predicted no antagonistic coactivation. The same model based on the mechanical stability criteria predicted 1.0% of maximum voluntary contraction coactivation of flexors and extensors with zero load and 3.1% of maximum voluntary contraction with a 32-kg mass. The stability model also was run with zero passive spine stiffness to simulate an injury. Under such conditions, the model predicted 3.4% and 5.5% of maximum voluntary contraction of antagonistic muscle coactivation for no extra load and the added 32 kg, respectively. CONCLUSIONS: This study demonstrated that antagonistic trunk flexor-extensor muscle coactivation was present around the neutral spine posture in healthy individuals. This coactivation increased with added mass to the torso. Using a biomechanical model, the coactivation was explained entirely on the basis of the need for the neuromuscular system to provide the mechanical stability to the lumbar spine.

Adult↗
PubMed1998

Reoperation in patients after anterior cervical plate stabilization in degenerative disease.

STUDY DESIGN: Consecutive case retrospective chart review. OBJECTIVES: First, to assess whether the number of' patients requiring a second cervical surgical intervention was changed as a result of using anterior cervical plate stabilization, and second, to determine the additional risks and/or benefits associated with the hardware implantation. SUMMARY OF BACKGROUND DATA: The optimal technique of performing stabilization, arthrodesis, and alignment of a cervical segment after discectomy with neural decompression in degenerative disease has yet to be determined. METHODS: The charts of 402 patients who had undergone an anterior cervical discectomy and arthrodesis for degenerative disease performed both with and without anterior cervical plate stabilization were reviewed, and reoperation data were compiled. The average follow-up time was 3.8 years (range, 1.5-9.4 years). RESULTS: Of 365 patients with 1- or 2-level cervical arthrodesis, 22 required a second surgical intervention (20 bone alone, 2 with anterior cervical plate stabilization). The Log-Rank test, which uses all patients and their total follow-up periods, was statistically significant favoring anterior cervical plate stabilization at one and two levels (P = 0.015). CONCLUSIONS: The addition of anterior cervical plate stabilization in one- and two-level cervical degenerative disease supplements the internal stabilization initially provided by the bone graft, and yields a lower reoperation rate.

Adult↗
PubMed1999

Stability of lumbar fusion with transpedicular fixation determined by roentgen stereophotogrammetric analysis.

STUDY DESIGN: An evaluation of the intervertebral stability of transpedicular instrumentation in posterolateral lumbar fusions by roentgen stereophotogrammetric analysis. OBJECTIVES: To determine the in vivo intervertebral stability of posterolateral lumbar fusions augmented with transpedicular screws and plates. SUMMARY OF BACKGROUND DATA: Transpedicular bone screw systems have been found to be as safe and clinically effective as other types of devices in stabilizing surgery of the spine. Many experimental studies have yielded basic data on the stabilizing implant effect in vitro, but the exact in vivo stabilizing effect on human lumbar vertebrae has not been presented previously. METHODS: In 12 patients, the intervertebral stability of posterolateral fusion in the lower lumbar spine augmented with transpedicular screws and plates was evaluated by serial roentgen stereophotogrammetric analysis with the patients in supine and erect positions 1 year after surgery. RESULTS: Screws in each fused vertebra yielded stable fixation or permitted sagittal intervertebral translations smaller than 1 mm induced by the positional change. A widely decompressed and destabilized vertebra without screw fixation yielded persisting intervertebral translations. CONCLUSIONS: The current study demonstrated the adequacy of in vivo stability of lumbar fusions augmented with transpedicular screws and plates. Sagittal translation seems easier to elicit than movements along the other three-dimensional axes. A widely decompressed and destabilized vertebra without screw fixation increases the risk for persisting intervertebral translations. The roentgen stereophotogrammetric analysis technique described seems to be a good way of comparing the in vivo behavior of different implant systems.

Adult↗
PubMed1999

Stability analysis of an enhanced load sharing posterior fixation device and its equivalent conventional device in a calf spine model.

STUDY DESIGN: An in vitro test of calf spine lumbar segments to compare biomechanical stabilization of a rigid versus a dynamic posterior fixation device. OBJECTIVES: To compare flexibility of a dynamic pedicle screw fixation device with an equivalent rigid device. SUMMARY OF BACKGROUND DATA: Dynamic pedicle screw device studies are not as prevalent in the literature as studies of rigid devices. These devices contain the potential to enhance load sharing and optimize fusion potential while maintaining stability similar to that of rigid systems. METHODS: Load-displacement tests were performed on intact and stabilized calf spines for the dynamic and rigid devices. Stability across a destabilized L3-L4 segment was restored by insertion of either a 6 mm x 40 mm dynamic or rigid pedicle screw fixation device across the L2-L4 segment. The screws then were removed, 7 mm x 45 mm pedicle screws of the opposite type were inserted, and the construct then was re-tested. Axial pull-out tests were performed to assess the likely effects of pedicle screw replacement on the load-displacement data. RESULTS: Results indicated a 65% reduction in motion in flexion-extension and a 90% reduction in lateral bending across the destabilized level for both devices, compared with intact spine values. Reduction in axial rotation motion was much smaller than in other modes. Axial pull-out tests showed no weakening of the bone-screw interface. CONCLUSIONS: Both devices provided significant stability of similar magnitudes in flexion, extension, and lateral bending. In axial rotation, the devices only could restore stability to levels similar to those in an intact spine. The dynamic device offers a design that may enhance load sharing without sacrificing construct stability.

Animals↗
PubMed1996

Regulation of melanoma cell adhesion stabilization to fibronectin.

Tumour cell arrest and the formation of stable adhesive interactions between tumour cells and endothelial cells or underlying matrix in the microvasculature are crucial steps in the metastatic process. We have developed a sensitive hydrodynamic adhesion assay to investigate the regulation of melanoma cell adhesion stabilization to the extracellular matrix protein fibronectin. Modulators of human MeWo melanoma Ca2+ concentration and stores, including ionomycin, thapsigargin, dantrolene and caffeine, inhibited cell adhesion stabilization to fibronectin; however, removal of Ca2+ from the extracellular medium did not affect stabilization. The calmodulin inhibitor W-7 and the protein kinase C inhibitor chelerythrine also blocked MeWo adhesion stabilization to fibronectin, as did the tyrosine kinase inhibitor genistein and the cytoskeletal inhibitor cytochalasin D. Manipulation of MeWo cell intracellular CAMP levels had no effect of adhesion stabilization to fibronectin, nor did treatment of cells with phorbol ester, pertussis toxin or cholera toxin. Drug treatments that inhibited adhesion stabilization also had significant effects on the actin cytoskeleton organization of the melanoma cells. This study suggests a role for calcium, calmodulin, protein kinase C and tyrosine kinases in the intracellular regulation of MeWo adhesive stabilization.

Actin Cytoskeleton↗
PubMed2004

Examination of static and dynamic postural stability in individuals with functionally stable and unstable ankles.

OBJECTIVE: To determine static and dynamic postural stability differences between functional ankle instability and stable ankle groups. DESIGN: Subjects were required to balance on a single leg and remain motionless for 20 seconds. After completing 3 trials, they performed a jump-landing test, which required them to jump 50% to 55% of their maximum vertical jump height. They landed on a single leg, stabilized quickly, and remained motionless for 20 seconds. SETTING: Sports Medicine Research Laboratory. PARTICIPANTS: Subjects with functional ankle instability (n = 14) who reported at least 2 sprains and "giving way" sensations at their ankle joint within the year prior to testing. Fourteen subjects with no history of ankle sprain injury were matched to subjects with functional ankle instability. MAIN OUTCOME MEASURES: Anterior/posterior and medial/lateral mean sway quantified static postural stability during single-leg stance. Dynamic postural stability was quantified with anterior/posterior and medial/lateral time to stabilization during single-leg jump landing. RESULTS: Mean sway was not significantly different between groups in the anterior/posterior (P = 0.28) and medial/lateral (P = 0.65) directions. The functional ankle instability group took significantly longer to stabilize in the anterior/posterior (3.27 +/- 0.72 seconds vs. 2.33 +/- 0.33 seconds; P < 0.001) and medial/lateral (2.48 +/- 0.50 seconds vs. 2.00 +/- 0.65 seconds; P = 0.04) directions. CONCLUSIONS: Individuals with functional ankle instability took significantly longer to stabilize than individuals with stable ankles after a single-leg jump landing. Differences between groups were not detected with mean sway measured during single-leg stance.

Adult↗
PubMed2005

Biomechanical stability of intramedullary nailed high proximal third tibial fractures with cement augmented proximal screws.

OBJECTIVE: Intramedullary nailing of nonarticular proximal tibia fractures can be affected by bone density resulting in loss of stability, fixation, and malalignment in osteopenic bone. This study was designed to quantify the biomechanical effects of augmenting proximal screws with cement in intramedullary nailing of high proximal third tibial fractures. DESIGN: In vitro biomechanical study using anatomic specimens. METHODS: Reamed nails were inserted into seven pairs of fresh-frozen cadaveric proximal tibiae and secured using two oblique and two transverse proximal screws. Paired tibiae were randomly assigned into two groups: cemented and noncemented proximal screw-holes. Bone cement was injected into the screw-holes before screw insertion in the cemented tibiae. Specimens were then tested in flexion/extension and varus/valgus to 12 Nm and in torsion to 7 Nm. Physical measurements of bone density were obtained to determine the effect of density on stability. MAIN OUTCOME MEASURES: Stability of the construct in both groups was analyzed and compared statistically using paired t tests. RESULTS: Cement augmentation of the proximal screws significantly increased mechanical stability in torsion and varus/valgus load configurations, with average decreases in rotational motion of 5.4 degrees +/- 1.6 degrees and 5.1 degrees +/- 5 degrees respectively. No change in stability was observed in flexion/extension loading. A trend toward decreased stability was seen in the uncemented construct in varus/valgus; cement augmentation of the proximal screws eliminated this effect. CONCLUSIONS: Lower bone density decreased the stability of the uncemented construct; however, cement augmentation of the proximal screws showed a trend to eliminate this effect in the varus/valgus loading configuration and should be considered when nailing proximal third tibial fractures in osteoporotic patients.

Aged↗
PubMed2003

Occipitocervical fusion for rheumatoid arthritis using the inside-outside stabilization technique.

STUDY DESIGN: A retrospective study investigating the clinical outcome of the inside-outside cranial bolt technique for occipitocervical stabilization used to manage rheumatoid arthritis was conducted. OBJECTIVE: To evaluate the safety and efficacy of the inside-outside technique for occipitocervical stabilization used to manage rheumatoid patients. SUMMARY OF BACKGROUND DATA: Achieving occipital cervical fusion for patients with rheumatoid arthritis is a complex and challenging problem. Complications related to placement of occipital screws have been reported. METHODS: Occipitocervical stabilization was used for atlantoaxial subluxation or basilar invagination in 21 patients with rheumatoid arthritis. The patients were assessed for pre- and postoperative neurologic status (Ranawat classification), neck pain, fusion and alignment, hardware complications, and continued posterior cranial settling. All the patients underwent stabilization using inside-outside occipital screws. The technique involves bilateral fixation of cervical plates to the occiput using inside-outside screws, and to the cervical spine using pars screws at C2 or lateral mass screws at subaxial levels. Bone grafting was accomplished with autologous rib or iliac crest graft. RESULTS: During the study, 14 women and 7 men with rheumatoid arthritis underwent occipitocervical stabilization and fusion. The average age of the patients was 65 years, and the mean follow-up period was 25.5 months. There were no surgical complications. Neck pain was reduced from an average Ranawat pain score of 2.40 to 0.4 (P < 0.0001). The Ranawat neurologic grade improved in 62% of the patients with preoperative neurologic deficit. Further cranial settling was not observed in any patient. There were no complications from implants and no incidence of instrumentation failure. CONCLUSIONS: The "inside-outside" technique is safe and effective for stabilizing the occipitocervical junction in rheumatoid patients. It is associated with significant reduction of neck pain, improved neurologic status, and maintenance of alignment and stability.

Aged↗
PubMed2004

The efficacy of a treatment program focusing on specific stabilizing exercises for pelvic girdle pain after pregnancy: a randomized controlled trial.

STUDY DESIGN: A randomized controlled trial with stratified block design. OBJECTIVES: To evaluate a treatment program focusing on whether specific stabilizing exercises for patients with pelvic girdle pain after pregnancy reduce pain, improve functional status, and improve quality of life. SUMMARY OF BACKGROUND DATA. : The evidence of effectiveness of treatment for pelvic girdle pain is weak. Recent research has focused on the importance of activation of muscles for motor control and stability of the lumbopelvic region. To the authors' knowledge, the efficacy of applying these principles for pelvic girdle pain has not previously been evaluated in a randomized controlled trial. METHODS: Eighty-one women with pelvic girdle pain were assigned randomly to two treatment groups for 20 weeks. One group received physical therapy with a focus on specific stabilizing exercises. The other group received individualized physical therapy without specific stabilizing exercises. Assessments were administered by a blinded assessor, at baseline, after intervention and 1 year post partum. Main outcome measures were pain, functional status and quality of life. RESULTS: There were no dropouts. After intervention and at 1 year post partum, the specific stabilizing exercise group showed statistically and clinically significant lower pain intensity, lower disability, and higher quality of life compared with the control group. Group difference in median values for evening pain after treatment was 30 mm on the Visual Analog Scale. Disability was reduced by more than 50% for the exercise group; changes were negligible in the control group. Significant differences were also observed for physical tests, in favor of the specific exercise group. CONCLUSION: An individualized treatment approach with specific stabilizing exercises appears to be more effective than physical therapy without specific stabilizing exercises for women with pelvic girdle pain after pregnancy.

Adult↗
PubMed2003

The evolution of population stability as a by-product of life-history evolution.

Proposed mechanisms for the evolution of population stability include group selection through longterm persistence, individual selection acting directly on stability determining the demographic parameters, and the evolution of stability as a by-product of life-history evolution. None of these hypotheses currently has clear empirical support. Using two sets of Drosophila melanogaster populations, we provide experimental evidence of stability evolving as a correlated response to selection on traits not directly related to demography. Four populations (FEJs) were selected for faster development and early reproduction for 125 generations, and the other four (JBs) were ancestral controls. All FEJ and JB populations have been maintained on discrete generations at moderate density, thus eliminating differential selection on stability determining demographic parameters. We derived eight small populations from each FEJ and JB population, and subjected four small populations each to either stabilizing or destabilizing food regimes. Census data on these 64 small populations over 20 generations clearly showed that the FEJ populations have significantly less temporal fluctuations in their numbers in both food regimes compared to their controls. This greater stability of the FEJ populations is probably a by-product of the evolution of reduced fecundity and pre-adult survivorship, as a correlated response to selection for rapid development.

Animals↗
PubMed2005

Nonparallel spatial stability of the boundary layer induced by Long's vortex on a solid plane perpendicular to its axis.

We consider the linear, viscous stability of the boundary layer induced by an unbounded vortex whose outer inviscid structure coincides near the axis with Long's vortex. The viscous boundary layer induced by the interaction of such a vortex with a solid plane perpendicular to the axis has a known self-similar structure. The spatial stability of this self-similar solution is analyzed here for axisymmetric and nonaxisymmetric perturbations propagating towards the axis of rotation. Viscous and nonparallel effects on the stability of the perturbations are retained up to their first order in the inverse of the local Reynolds number (nondimensional radius). The resulting parabolic stability equations are solved numerically using a spectral collocation method varying both the nondimensional frequency and radius. It is found that the flow is unstable to axisymmetric perturbations far away from the axis (inviscid instability). The growth rate of this inertial instability mode first increases and then decreases as the Reynolds number decreases (as the axis is approached). However, before this inviscid mode becomes stabilized, new viscous instabilities for both axisymmetric and nonaxisymmetric perturbations show up, which finally become stabilized at moderate Reynolds numbers. We characterize the critical Reynolds numbers and frequencies for the stability of these unstable perturbations as functions of their azimuthal wave number. It is found that the last perturbations that become stable as the axis is approached are nonaxisymmetric, corotating, perturbations with an azimuthal wave number n=4 .

Journal Article↗
PubMed2006

High-resolution structure of the p53 core domain: implications for binding small-molecule stabilizing compounds.

The p53 transcriptional regulator is the most frequently mutated protein in human cancers and the majority of tumor-derived p53 mutations map to the central DNA-binding core domain, with a subset of these mutations resulting in reduced p53 stability. Here, the 1.55 A crystal structure of the mouse p53 core domain with a molecule of tris(hydroxymethyl)aminomethane (Tris) bound through multiple hydrogen bonds to a region of p53 shown to be important for repair of a subset of tumor-derived p53-stability mutations is reported. Consistent with the hypothesis that Tris binding stabilizes the p53 core domain, equilibrium denaturation experiments are presented that demonstrate that Tris binding increases the thermodynamic stability of the mouse p53 core domain by 3.1 kJ mol(-1) and molecular-dynamic simulations are presented revealing an overall reduction in root-mean-square deviations of the core domain of 0.7 A when Tris is bound. It is also shown that these crystals of the p53 core domain are suitable for the multiple-solvent crystal structure approach to identify other potential binding sites for possible core-domain stabilization compounds. Analysis of the residue-specific temperature factors of the high-resolution core-domain structure, coupled with a comparison with other core-domain structures, also reveals that the L1, H1-S5 and S7-S8 core-domain loops, also shown to mediate various p53 activities, harbor inherent flexibility, suggesting that these regions might be targets for other p53-stabilizing compounds. Together, these studies provide a molecular scaffold for the structure-based design of p53-stabilization compounds for development as possible therapeutic agents.

2-Propanol↗
PubMed2006

Full-frame video stabilization with motion inpainting.

Video stabilization is an important video enhancement technology which aims at removing annoying shaky motion from videos. We propose a practical and robust approach of video stabilization that produces full-frame stabilized videos with good visual quality. While most previous methods end up with producing smaller size stabilized videos, our completion method can produce full-frame videos by naturally filling in missing image parts by locally aligning image data of neighboring frames. To achieve this, motion inpainting is proposed to enforce spatial and temporal consistency of the completion in both static and dynamic image areas. In addition, image quality in the stabilized video is enhanced with a new practical deblurring algorithm. Instead of estimating point spread functions, our method transfers and interpolates sharper image pixels of neighboring frames to increase the sharpness of the frame. The proposed video completion and deblurring methods enabled us to develop a complete video stabilizer which can naturally keep the original image quality in the stabilized videos. The effectiveness of our method is confirmed by extensive experiments over a wide variety of videos.

Algorithms↗
PubMed2006

Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of interleukin-4.

The role of disulfide bridges in the structure, stability, and folding pathways of proteins has been the subject of wide interest in the fields of protein design and engineering. However, the relative importance of entropic and enthalpic contributions for the stabilization of proteins provided by disulfides is not always clear. Here, we perform a detailed analysis of the role of disulfides in the conformational stability of human Interleukin-4 (IL4), a four-helix bundle protein. In order to evaluate the contribution of two out of the three disulfides to the structure and stability of IL4, two IL4 mutants, C3T-IL4 and C24T-IL4, were used. NMR and ANS binding experiments were compatible with altered dynamics and an increase of the nonpolar solvent-accessible surface area of the folded state of the mutant proteins. Chemical and thermal unfolding experiments followed by fluorescence and circular dichroism revealed that both mutant proteins have lower conformational stability than the wild-type protein. Transition temperatures of unfolding decreased 14 degrees C for C3T-IL4 and 10 degrees C for C24T-IL4, when compared to WT-IL4, and the conformational stability, at 25 degrees C, decreased 4.9 kcal/mol for C3T-IL4 and 3.2 kcal/mol for C24T-IL4. Interestingly, both the enthalpy and the entropy of unfolding, at the transition temperature, decreased in the mutant proteins. Moreover, a smaller change in heat capacity of unfolding was also observed for the mutants. Thus, disulfide bridges in IL4 play a critical role in maintaining the thermodynamic stability and core packing of the helix bundle.

Circular Dichroism↗
PubMed2006

Thermodynamic stability of a cold-adapted protein, type III antifreeze protein, and energetic contribution of salt bridges.

A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein (AFP), was carried out. The results indicate that the folding equilibrium of type III AFP is a reversible, unimolecular, two-state process with no populated intermediates. Compared to most mesophilic proteins whose folding is two-state, the psychrophilic type III AFP has a much lower thermodynamic stability at 25 degrees C, approximately 3 kcal/mol, and presents a remarkably downshifted stability-temperature curve, reaching a maximum of 5 kcal/mol around 0 degrees C. Type III AFPs contain few and non-optimally distributed surface charges relative to their mesophilic homologs, the C-terminal domains of sialic acid synthases. We used thermodynamic double mutant cycles to evaluate the energetic role of every surface salt bridge in type III AFP. Two isolated salt bridges provided no contribution to stability, while the Asp36-Arg39 salt bridge, involved in a salt bridge network with the C-terminal carboxylate, had a substantial contribution (approximately 1 kcal/mol). However, this contribution was more than counteracted by the destabilizing effect of the Asp36 carboxylate itself, whose removal led to a net 30% increase in stability at 25 degrees C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0 degrees C) at which AFPs must function. In addition, it indicates that salt bridge networks are used in nature also for the stability of psychrophilic proteins, and has led to a type III AFP variant of increased stability that could be used for biotechnological purposes.

Algorithms↗