Electromechanical properties of human osteoarthritic and asymptomatic articular cartilage are sensitive and early detectors of degeneration.

OBJECTIVE: To evaluate cross-correlations of ex vivo electromechanical properties with cartilage and subchondral bone plate thickness, as well as their sensitivity and specificity regarding early cartilage degeneration in human tibial plateau. METHOD: Six pairs of tibial plateaus were assessed ex vivo using an electromechanical probe (Arthro-BST) which measures a quantitative parameter (QP) reflecting articular cartilage compression-induced streaming potentials. Cartilage thickness was then measured with an automated thickness mapping technique using Mach-1 multiaxial mechanical tester. Subsequently, a visual assessment was performed by an experienced orthopedic surgeon using the International Cartilage Repair Society (ICRS) grading system. Each tibial plateau was finally evaluated with µCT scanner to determine the subchondral-bone plate thickness over the entire surface. RESULTS: Cross-correlations between assessments decreased with increasing degeneration level. Moreover, electromechanical QP and subchondral-bone plate thickness increased strongly with ICRS grade (ρ = 0.86 and ρ = 0.54 respectively), while cartilage thickness slightly increased (ρ = 0.27). Sensitivity and specificity analysis revealed that the electromechanical QP is the most performant to distinguish between different early degeneration stages, followed by subchondral-bone plate thickness and then cartilage thickness. Lastly, effect sizes of cartilage and subchondral-bone properties were established to evaluate whether cartilage or bone showed the most noticeable changes between normal (ICRS 0) and each early degenerative stage. Thus, the effect sizes of cartilage electromechanical QP were almost twice those of the subchondral-bone plate thickness, indicating greater sensitivity of electromechanical measurements to detect early osteoarthritis. CONCLUSION: The potential of electromechanical properties for the diagnosis of early human cartilage degeneration was highlighted and supported by cartilage thickness and µCT assessments.

Effect of topical corticosteroids on allergic airway inflammation and disease severity in obstructive sleep apnoea.

BACKGROUND: The incidence of sleep-related breathing disorders is correlated with lower and upper airway inflammatory diseases, such as asthma and allergic rhinitis. We hypothesized that corticosteroids treatment would lead to a greater reduction in disease severity in obstructive sleep apnoea syndrome (OSAS) patients with concomitant allergic rhinitis vs. non-allergic OSAS patients by reducing the level of inflammation in upper airway tissues. OBJECTIVE: This study was performed to determine whether treatment with intranasal corticosteroids could reduce upper airway inflammation and improve sleep parameters in obstructive sleep apnoea syndrome patients with or without concomitant allergic rhinitis. METHODS: Obstructive sleep apnoea syndrome patients with (n = 34) or without (n = 21) documented allergic rhinitis voluntarily enrolled in the study and were assessed at baseline and after corticosteroids treatment for 10-12 weeks. Sleep studies were performed and biopsies were obtained from the inferior turbinate, nasopharynx, and uvula. The apnoea-hypopnoea index, sleep quality, and level of daytime alertness were determined, and immunocytochemistry was used to phenotype tissue inflammation. RESULTS: Standard sleep indices improved following treatment in the entire cohort of obstructive sleep apnoea syndrome patients, with greater improvement seen in the allergic rhinitis group. Allergic rhinitis patients demonstrated significantly improved O2 saturation and a lower supine apnoea-hypopnoea index score after corticosteroid treatment; similar improvements were not seen in the non-allergic rhinitis group. Eosinophilia was detected at all three sites in the allergic rhinitis group, but not in the non-allergic rhinitis group. Following treatment, fewer eosinophils and CD4 lymphocytes were documented at all three biopsy sites in the allergic group; the reduction in inflammation was less apparent in the non-allergic rhinitis group. CONCLUSION: This study has provided important molecular and clinical evidence regarding the ability of corticosteroids to reduce upper airway inflammation and improve obstructive sleep apnoea syndrome morbidity patients with concomitant allergic rhinitis.

Electroarthrography: a novel method to assess articular cartilage and diagnose osteoarthritis by non-invasive measurement of load-induced electrical potentials at the surface of the knee.

OBJECTIVE: A new technique called electroarthrography (EAG) measures electrical potentials on the surface of the knee during joint loading. The objective of this study was to evaluate the effectiveness of EAG to assess joint cartilage degeneration. DESIGN: EAG recordings were performed on 20 asymptomatic subjects (Control group) and on 20 patients with bilateral knee osteoarthritis (OA) who had had a unilateral total knee replacement (TKR), both the TKR knee and the remaining knee were analyzed. EAG signals were recorded at eight electrode sites over one knee as the subjects shifted their weight from one leg to the other to achieve joint loading. The EAG signals were filtered, baseline-corrected and time-averaged. RESULTS: EAG repeatability was assessed with a test-retest protocol which showed statistically significant high intraclass correlation coefficients (ICC) for four electrode sites near the joint line. These sites also showed the highest mean EAG values. The mean EAG potentials of the Control group were significantly higher compared with the OA group for three sites overlying the joint line. The potentials overlying the TKR were statistically nul. In the Control group, no statistically significant correlation was found between the EAG amplitude and age, weight, height or body mass index (BMI); no statistical difference was found in mean EAG potentials between women and men. CONCLUSIONS: This study indicates that EAG signals arise from the streaming potentials in compressed articular cartilage which are known sensitive indicators of joint cartilage health. EAG is a promising new technique for the non-invasive assessment of cartilage degeneration and arthritis.

Humoral immunity 10 years after booster immunization with an adolescent and adult formulation combined tetanus, diphtheria, and 5-component acellular pertussis vaccine.

Persistence of antibodies after a single dose of Tdap vaccine (tetanus, diphtheria, and 5-component acellular pertussis vaccine) was evaluated in a follow-up study of adolescents (N=324) and adults (N=644) who had received Tdap in earlier clinical trials. Outcome measures were seroprotection (tetanus and diphtheria) or seropositivity (pertussis) and geometric mean concentrations. Humoral immune responses to all antigens were robust 1 month after initial immunization, decreased at subsequent measurements, but continued to exceed pre-immunization levels 1, 3, 5, and 10 years later. Protective levels of diphtheria and tetanus antitoxin persisted in 99.3% of adolescents 10 years after a booster dose of Tdap. Seropositivity to 1 or more pertussis antigens also persisted in most adolescents for 10 years. Although tetanus antitoxin responses were similar in adults to those observed in adolescents, diphtheria antitoxin titers were lower, reflecting the fact that a smaller proportion of adults had received diphtheria toxoid in the previous 10 years compared to adolescents. These data will contribute to the selection of the optimal interval for repeat doses of Tdap.

Angioplasty and stenting versus carotid-subclavian bypass for the treatment of isolated subclavian artery disease.

PURPOSE: To compare the results of a large series of percutaneous transluminal angioplasty (PTA)/stenting procedures in the subclavian artery with the results of a series of carotid-subclavian bypass grafts (CSBG) performed at the same institution for subclavian artery disease. METHODS: Between 1993 and 2006, 121 patients (43 men; mean age 63 years, range 38-85) underwent subclavian artery PTA/stenting and were compared to a group of 51 patients (29 men; mean age 62 years, range 46-75) with isolated subclavian artery occlusive disease treated with CSBG using polytetrafluoroethylene grafts. Graft or PTA/stenting patency was determined clinically and confirmed by Doppler pressures and/or duplex ultrasound/angiography. The cumulative patency and overall survival rates were calculated using the life-table method. RESULTS: The mean follow-up for the PTA/stent group was 3.4 years versus 7.7 years for the CSBG group. The technical success rate for the CSBG group was 100% versus 98% (119/121) for the PTA/stent group. The overall perioperative complication rate in the stent group was 15.1% (18/119: 11 minor and 7 major complications) versus 5.9% (3/51: 2 phrenic nerve palsy and 1 myocardial infarction) in the bypass group (p=0.093). There was no perioperative stroke or mortality in the CSBG group. The major perioperative complications in the stent group included 4 thromboembolic events, 1 congestive heart failure, 1 reperfusion arm edema, and 1 pseudoaneurysm. There was 1 perioperative death in the stent group. The 30-day patency rate was 100% for the bypass group and 97% (118/121) for the PTA/stent group. The primary patency rates at 1, 3, and 5 years were 100%, 98%, and 96% for the CSBG group versus 93%, 78%, and 70% for the stent group, respectively (p<0.0001). Freedom from symptom recurrence was also statistically superior in the bypass group versus the stent group (p<0.0001). There were no significant differences in the survival rates between both groups at any time point (p=0.322). CONCLUSION: Both CSBGs using PTFE grafts and subclavian PTA/stenting are safe, effective, and durable; however, CSBG is more durable in the long term. PTA/stenting of the subclavian artery should be the procedure of choice for high-risk patients; however, CSBG should be offered to good-risk surgical candidates who may be seeking a more durable procedure.

Temperature-dependent variations of ligand-receptor contact points in hAT(1).

Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protein-coupled receptors (GPCR). To this purpose benzophenone photolabels have been widely used to identify many contact residues in ligand-binding pockets. The three-dimensional binding environment of the human angiotensin II type 1 receptor hAT(1) has been determined using an iterative methionine mutagenesis strategy based on the photochemical properties and preferential incorporation of benzophenone onto methionine. This has led to the construction of a ligand-bound receptor structure. The present study investigated the effect of temperature on the accessibility of some of these contact points. The hAT(1) receptor and two representative Met mutants (H256M-hAT(1) and F293M-hAT(1)) from the iterative mutagenesis study were photolabelled with the benzophenone-ligand (125)I-[Sar(1), Bpa(8)]AngII at temperatures ranging from - 15 degrees C to 37 degrees C. Labelled receptors were partially purified and digested with cyanogen bromide to identify the contact points or segments. There were no changes in receptor contacts or labelling in the 7th transmembrane domains (TMD) of hAT(1) and F293M-hAT(1) across the temperature range. However, a temperature-dependent change in the ligand-receptor contact of H256M-hAT(1) was observed. At - 15 degrees C, H256M labelling was identical to that of hAT(1), indicating that the interaction was specific to the 7th TMD. Significant labelling changes were observed at higher temperatures and at 37 degrees C labelling occurred almost exclusively at mutated residue H256M-hAT(1) in the 6th TMD. Simultaneous competitive labelling of different areas of this target protein indicated that the ligand-receptor structure became increasingly fluctual at physiological temperatures, while a more compact, low mobility, and low energy conformation prevailed at low temperatures.

Similar success rates with bivalirudin and unfractionated heparin in bare-metal stent implantation.

BACKGROUND: Unfractionated heparin (UFH) is the traditional agent utilized during percutaneous peripheral interventions (PPIs) despite its well-known limitations. Bivalirudin, a thrombin-specific anticoagulant, overcomes many of the limitations of UFH and has consistently demonstrated comparable efficacy with significantly fewer bleeding complications. The purpose of this study was to compare procedural success in patients undergoing bare-metal stent implantation for atherosclerotic blockage of the renal, iliac, and femoral arteries and receiving either bivalirudin (0.75 mg/kg bolus/1.75 mg/kg/hr infusion) or UFH (50-70 U/kg/hr bolus) as the primary anticoagulant. METHODS: This study was an open-label, nonrandomized retrospective registry with the primary endpoint of procedural success. Secondary endpoints included incidence of: death, myocardial infarction (MI), urgent revascularization, amputation, and major and minor bleeding. RESULTS: One hundred and five consecutive patients were enrolled (bivalirudin = 53; heparin = 52). Baseline demographics were comparable between groups. Patients were pretreated with clopidogrel (approx. 71%) and aspirin (approx. 79%). Procedural success was achieved in 97% and 96% of patients in the bivalirudin- and heparin-treated groups, respectively. Event rates were low and similar between groups. CONCLUSION: Bivalirudin maintained an equal rate of procedural success in this cohort without sacrificing patient safety. Results of this study add to the growing body of evidence supporting the safety and efficacy of bivalirudin as a possible substitute for UFH in anticoagulation during peripheral vascular bare-metal stent implantation.

Evidence for two distinct pathways in TNFalpha-induced membrane and soluble forms of ICAM-1 in human osteoblast-like cells isolated from osteoarthritic patients.

OBJECTIVE: The present study aimed to investigate the modulation of membrane-bound intercellular adhesion molecule-1 (mICAM-1) and soluble ICAM-1 (sICAM-1) expression by tumor necrosis factor-alpha (TNFalpha) in human osteoarthritic (OA) osteoblasts. METHODS: Cultured human primary osteoblasts were stimulated with increasing concentrations of human recombinant TNFalpha. Expression of mICAM-1 and sICAM-1 was evaluated by immunocytochemistry, enzyme-linked immunosorbent assay and semi-quantitative reverse transcriptase-polymerase chain reaction. In addition, we investigated the molecular mechanisms underlying ICAM-1 induction by TNFalpha, focusing on the activation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways. RESULTS: Our data showed that TNFalpha dose-dependently increased mICAM-1 and sICAM-1 expression at the protein and mRNA levels in OA osteoblasts. The inhibitor of de novo mRNA synthesis, actinomycin D, suppressed TNFalpha-induced mICAM-1 and sICAM-1 expression. Upon examination of the signaling components, we found that TNFalpha was a potent activator of p38, p44/42, p54/46 MAPK, and IkappaBalpha (IkappaBalpha). The chemical inhibitors of p38, p44/42 MAPK, and NF-kappaB blocked TNFalpha-induced mICAM-1 expression but not that of sICAM-1. Transfection experiments revealed that p38 MAPK or IkappaB kinase alpha (IKKalpha) overexpression enhanced TNFalpha-induced mICAM-1 production. Furthermore, osteoblasts treatment with a chemical inhibitor of metalloproteinase-9 (MMP-9) activity, a proteolytic enzyme involved in ICAM-1 cleavage, evoked a significant 25% decrease of TNFalpha-induced sICAM-1 release. CONCLUSION: Taken together, these findings illustrate the central role played by TNFalpha in the regulation of ICAM-1. We suggest that TNFalpha differentially regulates sICAM-1 and mICAM-1 expression and that sICAM-1 release involves, in part, the proteolytic cleavage of mICAM-1 by MMP-9. The capacity of the MMP-9 inhibitor to prevent sICAM-1 production may be useful for the development of novel therapeutic approaches relevant to OA.

Periosteal autologous chondrocyte implantation for patellar chondral defect in patients with normal and abnormal patellar tracking.

Reported results for patellar ACI include a mixed population of patients having or not a concomitant correction of the extensor mechanism. No study has reported separate outcome for those two patellar ACI subgroups. Forty four patients were included in this study designed to compare the outcome of patellar ACI with extensor realignment (group A) to patellar ACI with normal patellofemoral tracking (group B). Group A (n=22) had a higher increase in average modified Cincinnati knee score (4.5 vs. 1.7 points), better function (1.7 vs. 2.5), better SF-36 physical component scores (70.9 vs. 55.4 points) and higher IKDC scores (85.2 vs. 60.6 points) when compared to group B (n=22) at an average follow-up of 2 years. Patellar ACI with concomitant extensor realignment has superior outcome than patellar ACI with normal patellofemoral tracking. An unloading osteotomy could be desirable in selected patients with normal patellofemoral biomechanics to improve the outcome in this population.

Subchondral and trabecular bone metabolism regulation in canine experimental knee osteoarthritis.

OBJECTIVE: To determine trabecular and subchondral bone metabolic changes in experimental canine osteoarthritis (OA). METHODS: OA was induced in 19 dogs by transection of the anterior cruciate ligament (ACL) of the right knee through a stab wound. Dogs were sacrificed at 8 (n=7) and 12 weeks (n=12) after surgery. Non-operated normal dogs (n=6) were used as controls. After sacrifice, samples were obtained from the weight-bearing area of medial tibial plateaus. Explants and cell cultures were prepared from subchondral and trabecular bone. Osteocalcin (Oc), cellular alkaline phosphatase (ALPase), urokinase plasminogen-activator (uPA), prostaglandin E2 (PGE2), metalloproteinase (MMP) and nitric oxide (NO) were measured using standard procedures. RESULTS: ALPase production was significantly increased only at week 12 in subchondral and trabecular bone, while an increase in Oc was noted at week 8. uPA and MMP activity were increased significantly at week 12 in subchondral bone, while PGE2 levels were significantly higher in subchondral and trabecular bone at week 12 compared to normal. A decrease in NO production appeared late at week 12 in trabecular bone, whereas NO levels from subchondral bone were significantly increased compared to normal at week 8. DISCUSSION: Intense bone remodeling takes place in both subchondral and trabecular bone in the knee following ACL transection. This process seems to occur around week 12, although Oc and NO appeared to be involved earlier at 8 weeks. These results suggest that not only subchondral but also trabecular bone metabolism is altered in this OA model.

Subclavian artery stenting: factors influencing long-term outcome.

This study provides extended follow-up of a nonrandomized series of symptomatic patients who underwent subclavian stent-supported angioplasty (SSA) with emphasis on preprocedure factors that may have influenced outcome. The endpoints of mortality and restenosis were analyzed using backward stepwise logistic regression with the following clinical variables: coronary artery disease, hypertension, hyperlipidemia, smoking, diabetes mellitus, chronic obstructive pulmonary disease, chronic renal insufficiency/failure, and hypothyroidism. Restenosis is reported based on prospective serial noninvasive studies and/or angiography. Mortality was evaluated by retrospective database review and inquiry to the State Department of Health and Human Services' statistical registry in patients who were lost to follow-up. Over a 9-year period (mean follow-up, 36.1 +/- 30.4 months; maximum observation, 109.5 months), 101 stents were placed in 91 consecutive patients (37 male, 54 female). The mean age at intervention was 62.03 +/- 9.3. The procedure was technically successful in 89 patients 97% (mean pre- and postoperative stenosis and pressure gradients were 90.2% +/- 9.4% vs. 3.7% +/- 6.6%, P < 0.001, and 59.9 +/- 35.2 vs. 0 mm Hg, P < 0.001, respectively), with 13 minor complications and no immediate major complications. One patient died of unrelated causes within 30 days. Per Kaplan-Meier method, for years 1 through 5, the rates of overall patency were 96%, 91%, 86%, 77%, and 72%; likewise, overall patient survival was 93%, 88%, 8%4, 81%, and 76%. No clear predictors for restenosis were discovered, although a trend toward higher recurrence was noted in women (18.5% in female vs. and 8.6% in male; P > 0.05), but the same were less likely to die during follow-up (P > 0.001). Also, the presence of hypothyroidism (P = 0.004) and increasing age (P = 0.068) were positively correlated with all-cause mortality. This study suggests that SSA is predictable, safe, and durable. The diagnosis of symptomatic subclavian disease is of prognostic importance, with age and male gender representing important predictors of all-cause long-term mortality. The strong association of increased mortality with hypothyroidism is difficult to discard and raises the question of a yet to be described thyroid steal phenomena.

Insights into steroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer in mitochondria: evidence from molecular modeling and structure-based thermodynamics supporting the existence of partially unfolded states of StAR.

The steroidogenic acute regulatory protein (StAR) is the major entrance for cholesterol in mitochondria under acute stimulation. Under such circumstances, dysfunctional StAR activity can ultimately lead to lipoid congenital adrenal hyperplasia (LCAH). A complete understanding of the StAR's molecular structure and mechanism is essential to comprehend LCAH. Thus far, there is no mechanistic model that can explain experimental results at the molecular level. This is partly due to the lack of the molecular structure of StAR. The closest approximation to the StAR molecular structure is the human MLN64 which has a similar activity to StAR, has a highly homologous primary structure and for which an X-ray structure is known. In this context, we have modeled the structure of StAR through standard homology modeling procedures based on the MLN64 structure. Our StAR model shows the presence of a hydrophobic cavity of 783.9 A(2) in surface area, large enough to fit one molecule of cholesterol. In addition, we have identified a unique charged pair, as in MLN64, lining the surface of the cavity and which could play a key role in the binding of cholesterol through the formation of an H-bond with its OH moiety. This suggests that the cholesterol-binding site of StAR is located inside this cavity. Taking into account that internal cavities are destabilizing to native protein structures and that the lining of the cavity has to become accessible in order to allow cholesterol binding, we have explored the possibility that StAR could exist in equilibrium with partially unfolded states. Using a structure-based thermodynamics approach, we show that partially folded states (with an unfolded C-terminal alpha-helix, and an open cavity) can be significantly populated at equilibrium and therefore allow cholesterol binding. These results are supported by recent experiments that show a loss of StAR helical character upon binding of an analog of cholesterol. Moreover, we show that the replacement of the residues involved in the charged-pair located in the binding site results in the loss of StAR activity, supporting a key role for these residues. Taken together, our results are applicable to StAR functioning both in the mitochondrial intermembrane space as well as outside the mitochondria.

Modulation of IL-1beta, IL-6, TNF-alpha and PGE(2) by pharmacological agents in explants of membranes from failed total hip replacement.

Introduction and goal Proinflammatory cytokines and prostaglandin E(2) (PGE(2)) play an important role in the pathophysiology of osteolysis and implant loosening. The aim of this study was to evaluate the role of pharmacological agents in the inhibition of Interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) and PGE(2) in explants of interface membranes from failed total hip replacements (fTHR). Material and methods Membranes from fTHR were retrieved (N=20) and explants were incubated for 72h in the absence or presence of tenidap at three different concentrations (5, 20 or 50 microg/ml) or diclofenac (125 microg/l). IL-1beta, IL-6, TNF-alpha, and PGE(2) levels were measured in the culture medium using ELISA Capture or EIA kits. Statistical analysis was done using the Mann-Whitney U-test. Results A statistically significant inhibition in IL-1beta synthesis was found at tenidap concentrations of 20 microg/ml (71.3%, P< 0.05) and 50 microg/ml (79.3%,P< 0.02). Tenidap reduced IL-6 levels by 90.4% at 20 microg/ml (P< 0.005) and 96.0% (P< 0.05) at 50 microg/ml. Tenidap also reduced the synthesis of TNF-alpha by 66.9% (P< 0.05) and 77.4% at concentrations of 20 microg/ml and 50 microg/ml. Tenidap had a marked suppressive effect of over 90% (P< 0.0001) on PGE(2) synthesis in all three concentrations. Diclofenac (125 microg/l) decreased PGE(2) production by 95% (P< 0.0001), but had no significant effect in IL-1beta, IL-6, and TNF-alpha levels in the culture medium. Conclusion The ability to simultaneously suppress the release of proinflammatory cytokines and PGE(2) may help control osteolysis and prevent aseptic loosening of THR. This effect could increase implant longevity and lead the way to the pharmacological treatment of this pathology.

Angiotensin II is bound to both receptors AT1 and AT2, parallel to the transmembrane domains and in an extended form.

UNLABELLED: We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. CONCLUSIONS: (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.

Temperature dependence of dynamics and thermodynamics of the regulatory domain of human cardiac troponin C.

Binding of Ca(2+) to the regulatory domain of troponin C (TnC) in cardiac muscle initiates a series of protein conformational changes and modified protein-protein interactions that initiate contraction. Cardiac TnC contains two Ca(2+) binding sites, with one site being naturally defunct. Previously, binding of Ca(2+) to the functional site in the regulatory domain of TnC was shown to lead to a decrease in conformational entropy (TDeltaS) of 2 and 0.5 kcal mol(-1) for the functional and nonfunctional sites, respectively, using (15)N nuclear magnetic resonance (NMR) relaxation studies [Spyracopoulos, L., et al. (1998) Biochemistry 37, 18032-18044]. In this study, backbone dynamics of the Ca(2+)-free regulatory domain are investigated by backbone amide (15)N relaxation measurements at eight temperatures from 5 to 45 degrees C. Analysis of the relaxation measurements yields an order parameter (S(2)) indicating the degree of spatial restriction for a backbone amide H-N vector. The temperature dependence of S(2) allows estimation of the contribution to protein heat capacity from pico- to nanosecond time scale conformational fluctuations on a per residue basis. The average heat capacity contribution (C(p,j)) from backbone conformational fluctuations for regions of secondary structure for the regulatory domain of cardiac apo-TnC is 6 cal mol(-1) K(-1). The average heat capacity for Ca(2+) binding site 1 is larger than that for site 2 by 1.3 +/- 0.8 cal mol(-1) K(-1), and likely represents a mechanism where differences in affinity between Ca(2+) binding sites for EF hand proteins can be modulated.

The contribution of arginine residues within the P6-P1 region of alpha 1-antitrypsin to its reaction with furin.

A series of mutants incorporating furin recognition sequences within the P6-P1 region of the reactive site loop of alpha(1)-antitrypsin were constructed. Variants containing different combinations of basic residues in the P1, P2, P4, and P6 positions replacing the wild type (P6)LEAIPM(P1) sequence were evaluated for their capacity to establish SDS-resistant complexes with furin, to affect association rate constants (k(ass) and k'(ass)), or to inhibit furin-dependent proteolysis of a model precursor in vivo. Each variant abolished processing of pro-von Willebrand factor in transfected hEK293 cells. The k(ass) of all variants were found to be similar (1.1-1.7 x 10(6) m(-1) s(-1)) except for one mutant, RERIRR, which had a k(ass) of 3.3 x 10(5) m(-1) s(-1). However, the stoichiometry of inhibition varied with values ranging from 2.9 to >24, indicating rapid formation of the acyl-enzyme intermediate (high k'(ass)). Moreover, those variants having high stoichiometry of inhibition values were accompanied by the rapid formation of cleaved forms of the inhibitors. The data suggest that the rate of conversion of the acyl-enzyme (EI') into the highly stable complex (EI*) was affected by replacement of specific residues within the reactive site loop. Taken together, the results reveal how furin recognition sequences within the context of the biochemical properties of serpins will play a role in the capacity of the protein to follow either the inhibitory or the substrate pathway.

Are trigger sequences essential in the folding of two-stranded alpha-helical coiled-coils?

The amino acid residues comprising the interface between strands of the coiled-coil motif are usually hydrophobic and make a major contribution to coiled-coil folding and stability. However, in some cases the presence of excellent hydrophobic residues at the coiled-coil interface is insufficient for folding. It has been proposed that a "consensus trigger sequence" exists that is necessary within the coiled-coil domains of various proteins to trigger folding. Therefore, in this study we designed a 31-residue hybrid sequence based on sequences from the two-stranded parallel coiled-coil domains of the yeast transcriptional activator GCN4 and the actin-bundling protein Dictyostelium discoideum cortexillin I. The hybrid and its analogs were studied by CD spectroscopy and analytical ultracentrifugation. The hybrid had stable residues in the core "a" and "d" positions in the 3-4 hydrophobic repeat, denoted (abcdefg)n, but did not have a consensus trigger sequence and did not possess appreciable secondary structure as determined by CD spectroscopy. The substitutions in the parent peptide were introduced at positions other than "a" and "d", altering a variety of interactions including alpha-helical propensity, interchain and intrachain electrostatics, and hydrophobicity. Although the substitutions did not bring the overall sequence in closer agreement to the consensus trigger sequence, they increased coiled-coil folding and stability. Therefore, our results suggest that the combination of stabilizing effects along a protein sequence is a more general indicator of protein folding in coiled-coils than the identification of a specific trigger sequence. We propose that surpassing a critical threshold stability value using any type or combination of stabilizing effects will allow coiled-coils to fold, in the absence of a specific trigger sequence per se.

Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position "d".

We describe the de novo design and biophysical characterization of a model coiled-coil protein in which we have systematically substituted 20 different amino acid residues in the central "d" position. The model protein consists of two identical 38 residue polypeptide chains covalently linked at their N termini via a disulfide bridge. The hydrophobic core contained Val and Ile residues at positions "a" and Leu residues at positions "d". This core allowed for the formation of both two-stranded and three-stranded coiled-coils in benign buffer, depending on the substitution at position "d". The structure of each analog was analyzed by CD spectroscopy and their relative stability determined by chemical denaturation using GdnHCI (all analogs denatured from the two-stranded state). The oligomeric state(s) was determined by high-performance size-exclusion chromatography and sedimentation equilibrium analysis in benign medium. Our results showed a thermodynamic stability order (in order of decreasing stability) of: Leu, Met, Ile, Tyr, Phe, Val, Gln, Ala, Trp, Asn, His, Thr, Lys, Ser, Asp, Glu, Arg, Orn, and Gly. The Pro analog prevented coiled-coil formation. The overall stability range was 7.4 kcal/mol from the lowest to the highest analog, indicating the importance of the hydrophobic core and the dramatic effect a single substitution in the core can have upon the stability of the protein fold. In general, the side-chain contribution to the level of stability correlated with side-chain hydrophobicity. Molecular modelling studies, however, showed that packing effects could explain deviations from a direct correlation. In regards to oligomerization state, eight analogs demonstrated the ability to populate exclusively one oligomerization state in benign buffer (0.1 M KCl, 0.05 M K(2)PO(4)(pH 7)). Ile and Val (the beta-branched residues) induced the three-stranded oligomerization state, whereas Tyr, Lys, Arg, Orn, Glu and Asp induced the two-stranded state. Asn, Gln, Ser, Ala, Gly, Phe, Leu, Met and Trp analogs were indiscriminate and populated two-stranded and three-stranded states. Comparison of these results with similar substitutions in position "a" highlights the positional effects of individual residues in defining the stability and numbers of polypeptide chains occurring in a coiled-coil structure. Overall, these results in conjunction with other work now generate a relative thermodynamic stability scale for 19 naturally occurring amino acid residues in either an "a" or "d" position of a two-stranded coiled-coil. Thus, these results will aid in the de novo design of new coiled-coil structures, a better understanding of their structure/function relationships and the design of algorithms to predict the presence of coiled-coils within native protein sequences.