Investigation of signal transduction routes within the sensor/transducer protein BlaR1 of Staphylococcus aureus.

The transmembrane antibiotic sensor/signal transducer protein BlaR1 is part of a cohort of proteins that confer ß-lactam antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) [Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) Chem. Rev. 105, 395-424; Llarrull, L. I., Fisher, J. F., and Mobashery, S. (2009) Antimicrob. Agents Chemother. 53, 4051-4063; Llarrull, L. I., Toth, M., Champion, M. M., and Mobashery, S. (2011) J. Biol. Chem. 286, 38148-38158]. Specifically, BlaR1 regulates the inducible expression of ß-lactamases that hydrolytically destroy ß-lactam antibiotics. The resistance phenotype starts with ß-lactam antibiotic acylation of the BlaR1 extracellular domain (BlaRS). The acylation activates the cytoplasmic protease domain through an obscure signal transduction mechanism. Here, we compare protein dynamics of apo versus antibiotic-acylated BlaRS using nuclear magnetic resonance. Our analyses reveal inter-residue interactions that relay acylation-induced perturbations within the antibiotic-binding site to the transmembrane helix regions near the membrane surface. These are the first insights into the process of signal transduction by BlaR1.

Structural characterization of the caveolin scaffolding domain in association with cholesterol-rich membranes.

Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a ß-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect ß-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization.

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions.

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular ß-sheet contacts present in the monomeric state could constitute intermolecular ß-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded ß-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register ß-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular ß-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.

The aggregation-enhancing huntingtin N-terminus is helical in amyloid fibrils.

The 17-residue N-terminus (htt(NT)) directly flanking the polyQ sequence in huntingtin (htt) N-terminal fragments plays a crucial role in initiating and accelerating the aggregation process that is associated with Huntington's disease pathogenesis. Here we report on magic-angle-spinning solid-state NMR studies of the amyloid-like aggregates of an htt N-terminal fragment. We find that the polyQ portion of this peptide exists in a rigid, dehydrated amyloid core that is structurally similar to simpler polyQ fibrils and may contain antiparallel ß-sheets. In contrast, the htt(NT) sequence in the aggregates is composed in part of a well-defined helix, which likely also exists in early oligomeric aggregates. Further NMR experiments demonstrate that the N-terminal helical segment displays increased dynamics and water exposure. Given its specific contribution to the initiation, rate, and mechanism of fibril formation, the helical nature of htt(NT) and its apparent lack of effect on the polyQ fibril core structure seem surprising. The results provide new details about these disease-associated aggregates and also provide a clear example of an amino acid sequence that greatly enhances the rate of amyloid formation while itself not taking part in the amyloid structure. There is an interesting mechanistic analogy to recent reports pointing out the early-stage contributions of transient intermolecular helix-helix interactions in the aggregation behavior of various other amyloid fibrils.

Phosphatidylcholine "wobble" in vesicles assessed by high-resolution 13C field cycling NMR spectroscopy.

High resolution (13)C NMR field cycling (covering 11.7 down to 0.002 T) relaxation studies of the sn-2 carbonyl of phosphatidylcholines in vesicles provide a detailed look at the dynamics of this position of the phospholipid in vesicles. The spin-lattice relaxation rate, R(1), observed down to 0.05 T is the result of dipolar and CSA relaxation components characterized by a single correlation time tau(c), with a small contribution from a faster motion contributing to CSA relaxation. At lower fields, R(1) increases further with a correlation time consistent with vesicle tumbling. The tau(c) is particularly interesting since it is 2-3 times slower than what is observed for (31)P of the same phospholipid. However, cholesterol increases the tau(c) for both (31)P and (13)C sites to the same value, approximately 25 ns. These observations suggest faster local motion dominates the dipolar relaxation of the (31)P, while a slower rotation or wobble dominates the relaxation of the carbonyl carbon by the alpha-CH(2) group. The faster motion must be damped with the sterol present. As a general methodology, high resolution (13)C field cycling may be useful for quantifying dynamics in other complex systems as long as a (13)C label (without attached protons) can be introduced.

The long-term effect of successful mitral balloon valvotomy on left atrial size.

BACKGROUND AND AIM OF THE STUDY: The study aim was to determine the extent of regression of left atrial (LA) enlargement following mitral balloon valvotomy (MBV) for mitral stenosis. METHODS: Data obtained from 205 patients before, and at a mean of 31.0 +/- 21.1 months (range: 6 to 86.3 months) after successful MBV were analyzed retrospectively. RESULTS: The invasively determined mitral valve area increased from 0.81 +/- 0.27 cm2 at baseline to 1.73 +/- 0.54 cm2 immediately after valvotomy (p <0.0001), and the mean mitral gradient fell from 15.6 +/- 5.3 to 5.4 +/- 2.5 mmHg (p <0.0001). Similar changes were noted in Doppler-determined mitral valve area (0.89 +/- 0.16 to 1.97 +/- 0.29 cm2; p <0.0001) and gradient (12.6 +/- 5.3 to 4.9 +/- 1.7 mmHg; p <0.0001). In comparison with baseline, significant (p <0.0001) reductions were noted at follow up in the echocardiographic anteroposterior (48.7 +/- 6.9 to 42.4 +/- 6.6 mm), superior-inferior (68.5 +/-8.1 to 59.6 +/- 8.2 mm) and medial-lateral LA dimension (51.2 +/- 6.7 to 44.1 +/- 7.7 mm) and calculated LA volume (91.6 +/- 29.1 to 60.7 +/- 23.8 cm3) Patients in atrial fibrillation had larger LA dimensions, but substantially smaller absolute and relative reduction in LA size at follow up than patients in sinus rhythm. Among patients with prevalvotomy LA enlargement, normalization of LA dimension at follow up was seen in 29.2% of patients in sinus rhythm, but in none of the 32 with atrial fibrillation. CONCLUSIONS: Successful MBV results in significant long-term reduction in LA size in most patients, but normalization of LA size is unusual.

Long-term effects of balloon angioplasty on systemic hypertension in adolescent and adult patients with coarctation of the aorta.

AIMS: To define the long-term effect of balloon angioplasty of aortic coarctation on hypertension, in adolescent and adult patients. METHODS: Balloon angioplasty of discrete, native aortic coarctation was performed on 50 patients (34 male) aged 23+/-8 (mean+/-standard deviation) years. In 42 of these patients cardiac catheterization and angiography were repeated 1 year later, and on the basis of sphygmomanometric blood pressure determination at that time, they were divided into 31 patients (group A) with normalized blood pressure and 11 patients (group B) who still needed antihypertensive medication. Both groups were followed annually thereafter for 12-123 (66+/-37) months. RESULTS: Coarctation gradient values before, immediately after and 1 year after angioplasty were 69+/-24 mmHg, 12+/-8 mmHg (P<0.001) and 7+/-6 mmHg. The corresponding systolic blood pressure values were 165+/-17 mmHg, 128+/-12 mmHg (P<0.001) and 115+/-10 mmHg (P<0.001) in group A; 182+/-21 mmHg, 141+/-24 mmHg (P<0.001) and 134+/-18 mmHg (P<0.001) in group B. Echocardiographic left ventricular mass index before angioplasty and at follow-up was 130+/-31 g x m-2 and 105+/-23 g x m-2 in group A; 157+/-38 g x m-2 and 132+/-35 g x m-2 in group B (P<0.001 for both comparisons). CONCLUSION: Normalization of blood pressure without medication occurred in 74% of patients after angioplasty for aortic coarctation, with subsequent long-term regression of left ventricular hypertrophy. In comparison to reported surgical results, balloon angioplasty should be considered as first line treatment for native, discrete aortic coarctation in adolescent and adult patients.

The role of selenium deficiency in dilated cardiomyopathy in Saudi Arabia.

BACKGROUND: Selenium deficiency is implicated in the etiology of endemic juvenile dilated cardiomyopathy in China, and in sporadic cases in other countries. The aim of this study was to evaluate the role of selenium deficiency in the pathophysiology of dilated cardiomyopathy in the Saudi Arabian population. PATIENTS AND METHODS: Plasma and urine selenium concentrations from 72 Saudi patients with confirmed dilated cardiomyopathy were compared with corresponding values from 70 control subjects of the same national origin who had normal ventricular function. RESULTS: Plasma and urine selenium concentrations (mean+/-SD) were 1.347plusmn;0.45 and 0.49+/-0.37 micromol/L, respectively, for the patient group, and 1.32+/-0.41 and 0.60+/-0.41 micromol/L, respectively, for the control group. The differences in the values between the two groups were statistically insignificant. CONCLUSION: In the Saudi population, dilated cardiomyopathy is not caused by selenium deficiency.

One- to ten-year follow-up results of balloon angioplasty of native coarctation of the aorta in adolescents and adults.

OBJECTIVES: We attempted to evaluate the role of balloon angioplasty in the treatment of discrete coarctation of the aorta in adolescents and adults, with special emphasis on long-term results. BACKGROUND: Controversy persists over the use of balloon dilation for the treatment of native coarctation of the aorta. METHODS: Between July 1986 and January 1997, 43 consecutive adolescent and adult patients with discrete coarctation of the aorta underwent balloon angioplasty. One- to 10-year follow-up data of 37 patients, including results of cardiac catheterization and magnetic resonance imaging (MRI), form the basis of this study. RESULTS: No early or late deaths occurred. Balloon angioplasty produced a reduction in the peak to peak coarctation gradient from a mean +/- SD of 69 +/- 24 mm Hg (95% confidence interval [CI] 61 to 76) to 12 +/- 8 mm Hg (95% CI 10 to 14.8) (p < 0.001). Follow-up catheterization 12 months later (37 patients) revealed a residual gradient of 6.7 +/- 6 mm Hg (95% CI 4.6 to 8.9); 3 (7%) of 43 patients had suboptimal results with development of recoarctation, defined as peak gradient >20 mm Hg, with successful repeat angioplasty. A small aneurysm developed at the site of dilation in 3 (7%) of the 43 patients. MRI follow-up data 1 to 10.8 years (mean 5.2 +/- 2.7) after angioplasty (37 patients) revealed no new aneurysm or appreciable change in the size of the preexisting aneurysm in the three patients. The blood pressure had normalized without medication in 27 (73%) of 37 patients at follow-up examination. CONCLUSIONS: Balloon angioplasty is safe and effective and should be considered a viable alternative to operation for treatment of discrete coarctation of the aorta in adolescents and adults.

Immediate and long-term effect of mitral balloon valvotomy on left ventricular volume and systolic function in severe mitral stenosis.

To determine the immediate and long-term effect of mitral balloon valvotomy (MBV) on left ventricular (LV) volume and function, we studied 17 patients (mean age 27 +/- 9 years) with severe mitral stenosis undergoing MBV by cardiac catheterization and angiography before and immediately after MBV and at mean 12 months later. At baseline, LV end-diastolic volume index (EDVI) was reduced. Ten patients had EDVI < or = 55 ml/m2, and four patients (23.5%) had LV ejection fraction < 50%. EDVI increased from 60 +/- 17 ml/m2 to 66 +/- 17 ml/m2 (p < 0.05) immediately after MBV and increased further to 72 +/- 16 ml/m2 (p < 0.05) later. Stroke volume index increased from 34 +/- 10 ml/m2 to 41 +/- 12 ml/m2 (p < 0.05) immediately after MBV and increased further to 50 +/- 11 ml/m2 (p < 0.001) later. LV end diastolic pressure increased from 12 +/- 5 mm HG to 16 +/- 4 mm HG (p < 0.05) immediately after MBV and fell to 13 +/- Hg at follow-up. LV ejection fraction increased from 57 +/- 7% to 62 +/- 6% (p < 0.05) immediately after MBV and 71 +/- 8% later (p < 0.001). Mean systolic ejection rate increased from 82 +/- 35 ml/sec to 101 +/- 48 ml/sec (p < 0.05) immediately after and 165 +/- 81 ml/sec later (p < 0.05). Systemic vascular resistance fell from 1887 +/- 525 dyne/sec/cm-5 to 1280 +/- 231 dyne/sec/cm-5 (p < 0.001) at follow-up. We conclude that the LV end-diastolic volume and systolic function are reduced in patients with mitral stenosis, and the LV end-diastolic volume is increased immediately after MBV and continues to increase at follow-up 12 months later; the LV ejection performance improves after successful MBV because of an increase in end-diastolic LV volume (preload) and reduction of SVR.

Advantage of Inoue balloon catheter in mitral balloon valvotomy: experience with 220 consecutive patients.

Percutaneous mitral balloon valvotomy (PMV) using the Inoue balloon catheter was attempted in 220 consecutive patients with severe symptomatic mitral stenosis. Their age range was 10-63 mean 30 +/- 10 years; 161 patients were females and 59 were males; 29 patients were in atrial fibrillation. Eleven patients were pregnant; 14 patients underwent previous surgical commissurotomy. The procedure was technically successfully performed in 215 (97.7%) patients. The mean fluoroscopy time was 15.5 +/- 6.4 min and mean procedure time was 109 +/- 79 min. Optimal results (group I) was achieved in 207 patients who have mitral score of 7 +/- 1. PMV resulted in decrease in left atrial pressure from 23 +/- 5 to 14 +/- 4 mm Hg (P < 0.001), the mean mitral valve gradient (MVG) decreased from 15 +/- 4 to 6 +/- 3 mm Hg (P < 0.001). The mitral valve area (MVA) by catheter increased from 0.7 +/- 0.2 to 1.7 +/- 0.5 cm2 (P < 0.001) and MVA as determined by echocardiography (2DE) increased from 0.8 +/- 0.1 to 1.9 +/- 0.3 cm2 (P < 0.001). The results were suboptimal in eight patients who have a mitral score of 10 +/- 1 (group II) MVA by catheter increased from 0.7 +/- 0.2 to 1 +/- 0.1 cm2 and Doppler MVA increased from 0.8 +/- 0.1 to 1.3 +/- 0.1 cm2. There were no deaths or thromboembolism. Two patients developed cardiac tamponade; mild mitral regurgitation (MR) developed in 24 patients (11%) and increased by one grade in another 22 patients (10%). Severe MR was encountered in three patients (1.4%). A small ASD (QP/QS < or = 1.3) was detected by oximetry in 5% of patients and by color-flow mapping in 26% of patients. One hundred fifty-eight patients from group I were followed up, for a mean of 32 +/- 12 months; MVA remained at 1.7 +/- 0.4 cm2. Seven patients developed mitral restenosis in group I, and 5 out of 8 patients developed restenosis in group II. We conclude that the hemodynamic results are good and comparable with those reported with double balloon technique. However, the Inoue balloon has several advantages over the double balloon technique: (1) low incidence of mitral regurgitation and ASDs; (2) shorter procedure and fluoroscopy time; and (3) low complication rates and the valve anatomy affects the immediate and late outcome of mitral balloon valvotomy.

Immediate and long-term effect of mitral balloon valvotomy on severe pulmonary hypertension in patients with mitral stenosis.

The pulmonary vascular hemodynamics were studied in 21 patients with severe mitral stenosis and severe pulmonary hypertension. Hemodynamic data were obtained before and immediately after mitral balloon valvotomy (MBV) and at follow-up 7 to 14 months (mean 12 months) later by repeat catheterization. The mean pulmonary capillary wedge pressure (PCW) decreased from 27 +/- 5 to 15 +/- 4 mm Hg (p < 0.001). The mean mitral valve gradient (MVG) decreased from 18 +/- 4 to 6 +/- 2 mm Hg (p < 0.001). Mitral valve area (MVA) increased from 0.6 +/- 0.1 to 1.5 +/- 0.3 cm2 (p < 0.02). Cardiac index increased from 2.2 +/- 0.3 to 2.6 to 0.5 L/min/m2 (p < 0.02). The pulmonary artery systolic pressure decreased from 65 +/- 13 to 50 +/- 13 mm Hg (p < 0.001), and no significant change was seen in pulmonary vascular resistance (PVR) immediately after MBV from 461 +/- 149 to 401 +/- 227 dynes/sec/cm(-5) (p = 0.02). At follow-up the MVA increased from 1.5 +/- 0.3 to 1.7 +/- 0.3 cm2 (p < 0.02). Cardiac index increased further to 3 +/- 0.4 L/min/m2 (p < 0.02). MVG and PCW pressure remained the same. The pulmonary artery systolic pressure decreased further to 38 +/- 9 mm Hg (p < 0.02). PVR decreased significantly to 212 +/- 99 dynes/sec/cm(-5) (p < 0.02). We concluded that the pulmonary artery pressure decreased without normalizing immediately after MBV and normalized in patients with optimal results from mitral balloon valvotomy 7 to 14 months later. Insignificant change in PVR was seen immediately after MBV and markedly decreased or normalized at late follow-up in patients with optimal result from MBV.

Endomyocardial fibrosis: Clinical, echocardiographic and hemodynamic findings.

Endomyocardial fibrosis (EMF) is a disease of unknown origin. It was first described by Davies in Uganda in 1948. The clinical enchocardiographic, and hemodynamic findings in 18 patients are presented. Six patients had right-sided involvement, four had left-sided involvement and eight had biventricular involvement. The presence of a small ventricle with obliteration of the apex and a large atrium, diagnosed by two-dimensional echocardiography, is highly suggestive of endomyocardial fibrosis. Ventricular angiography was diagnostic in 17 out of 18 cases. Endomyocardial biopsy yielded positive findings in three out of seven patients and is not essential for diagnosis. Two patients suffered a cerebral embolism. Six patients underwent surgery with good results in three patients.