The structure of the mite allergen Blo t 1 explains the limited antibody cross-reactivity to Der p 1.

The Blomia tropicalis (Blo t) mite species is considered a storage mite in temperate climate zones and an important source of indoor allergens causing allergic asthma and rhinitis in tropical and subtropical regions. Here, we report the crystal structure of one of the allergens from Blo t, recombinant proBlo t 1 (rproBlo t 1), determined at 2.1 Å resolution. Overall, the fold of rproBlo t 1 is characteristic for the pro-form of cysteine proteases from the C1A class. Structural comparison of experimentally mapped Der f 1/Der p1 IgG epitopes to the same surface patch on Blo t 1, as well as of sequence identity of surface-exposed residues, suggests limited cross-reactivity between these allergens and Blo t 1. This is in agreement with ELISA inhibition results showing that, although cross-reactive human IgE epitopes exist, there are unique IgE epitopes for both Blo t 1 and Der p 1.

Measuring myocardial perfusion: the role of PET, MRI and CT.

Recently, focus has changed from anatomical assessment of coronary arteries towards functional testing to evaluate the effect of stenosis on the myocardium before intervention. Besides positron-emission tomography (PET), cardiac MRI (CMR), and cardiac CT are able to measure myocardial perfusion. Myocardial perfusion abnormalities are the first sign of the ischaemic cascade in the development of coronary artery disease (CAD). PET is considered the non-invasive clinical reference standard for absolute quantification of myocardial perfusion. The diagnostic and prognostic value of PET is well-known and is used in routine clinical practice. However, PET uses radioactive tracers and has a lower spatial resolution compared to CMR and CT. CMR and CT are emerging techniques in the field of myocardial perfusion imaging. CMR uses magnetic resonance to obtain images, whereas CT uses x-rays during first-pass of non-ionic and ionic contrast agents, respectively. Absolute quantification with CMR has yet to be established in routine clinical practice, while CT has yet to prove its diagnostic and prognostic value. The upcoming years may change the way we diagnose and treat patients suspected of having CAD with more precise methods for measuring myocardial perfusion. The aim of this comprehensive review is to discuss current and emerging imaging techniques used for myocardial perfusion imaging.

The inflammatory biomarker YKL-40 at admission is a strong predictor of overall mortality.

OBJECTIVES: YKL-40 is an inflammatory biomarker associated with disease activity and mortality in patients with diseases characterized by inflammation and tissue remodelling. The aim of this study was to describe the prognostic value of YKL-40 in an unselected patient population. DESIGN: In consecutive patients admitted to hospital during a 1-year period, blood was collected and information regarding final diagnosis and mortality was collected. Median follow-up time was 11.5 years. SETTING: District hospital, Copenhagen, Denmark. PATIENTS: A total of 1407 patients >40 years of age were admitted acutely. MAIN OUTCOME MEASURE: All-cause mortality. RESULTS: Median YKL-40 was increased in patients (157 µg L(-1) , range 13-7704 µg L(-1) ) compared to healthy controls (40 µg L(-1) , range 29-58 µg L(-1) ; P < 0.001). Patients with YKL-40 in the highest quartile had a hazard ratio (HR) of 7.1 [95% confidence interval (CI) 4.2-12.0] for all-cause mortality in the first year and 3.4 (95% CI 2.8-4.2) in the total study period, compared to those in the lowest quartile (HR = 1). The HR for death for all patients with YKL-40 above the normal age-corrected 95th percentile was 2.1 (95% CI 1.6-2.7) after 1 year and 1.5 (95% CI 1.3-1.7) during the total study period, compared to patients with YKL-40 below the age-corrected 95th percentile. The results of multivariable analysis showed that YKL-40 was an independent biomarker of mortality; this was most significant in the first year. YKL-40 was a marker of prognosis in all disease categories. The HR for death was increased in patients with YKL-40 above the normal age-corrected 95th percentile in healthy subjects independent of type of disease (all P < 0.001). CONCLUSION: The level of YKL-40 at admission is a strong predictor of overall mortality, independent of diagnosis and could be useful as a biomarker in the acute evaluation of all patients.

Regulation and function of the CD3gamma DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro.

Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3gamma. Thus, phosphorylation of CD3gamma serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3gamma internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3gamma chimeras; and in vitro by binding CD3gamma peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3gamma D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3gamma molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3gamma S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.

beta2-adaptin is constitutively de-phosphorylated by serine/threonine protein phosphatase PP2A and phosphorylated by a staurosporine-sensitive kinase.

Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated that phosphorylated AP2 complexes were found more evenly distributed at the plasma membrane compared to non-phosphorylated AP2 complexes which were found in aggregates. Finally, we found that phosphorylation of beta2-adaptin correlated with inhibition of clathrin-mediated endocytosis. Our results support the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.

Plasma disappearance of albumin and impact of capillary thickness in idiopathic dilated cardiomyopathy and after heart transplantation.

BACKGROUND-The increased plasma disappearance of albumin has previously been described in decompensated congestive heart failure (CHF); this disappearance normalized after diuretic treatment. Cardiac transplantation (HTX) and current medical treatment affect microvascular structure and function. We investigated the plasma disappearance of albumin and the impact of microvascular thickness and electrostatic properties in patients with compensated CHF and after HTX. METHODS AND RESULTS-The fraction of intravascular albumin that passes to the extravascular space per unit time, as determined from the plasma disappearance of intravenously injected (131)I-labeled albumin, was increased to 7.8+/-1.7% in 16 patients with CHF compared with 18 controls (6.5+/-1.9%, P<0.05); these levels normalized after HTX (5.8+/-2.6%, P<0.01, n=17). The change in ratio between (131)I-albumin and simultaneously injected negatively charged glycosylated (125)I-albumin (selectivity index, >1/hour in controls) was lower in patients with HTX (0.993+/-0. 022/hour) than in controls (1.008+/-0.019/hour; P<0.05), which indicated a relatively increased plasma disappearance of negatively charged albumin in HTX patients. Capillary basement membrane thickness was evaluated semiquantitatively from skin biopsies and showed no difference in the 3 groups (control, CHF, and HTX patients). However, in all 3 study groups, subjects with thicker capillary basement membranes had lower albumin escape rates (6.1+/-1. 8%, n=32, versus 7.6+/-2.6% in subjects without thickening of capillary basement membranes, n=19; P<0.05). CONCLUSIONS-The plasma disappearance of albumin increased in patients with compensated CHF and it normalized after HTX. The present normalized capillary basement thicknesses in patients with CHF and the direct association between this parameter and plasma albumin disappearance indicate that previous compensatory microvascular basement membrane growth results in restricted permeability. Microvascular electrostatic properties did not relate to plasma albumin disappearance.

Altered peripheral vasodilator profile of nitroglycerin during long-term infusion of N-acetylcysteine.

OBJECTIVES: The aim of this study was to compare the short- and long-term effects of intravenous nitroglycerin plus placebo and nitroglycerin plus N-acetylcysteine on peripheral arteries, veins and microcirculation in humans. BACKGROUND: The thiol donor N-acetylcysteine may potentiate the hemodynamic response to nitrates in nitrate-tolerant and nontolerant patients. The vascular changes responsible for this effect are not clear. METHODS: Eight male volunteers were treated with nitroglycerin (0.1 microgram/kg per min) combined with N-acetylcysteine (2 g intravenously, followed by 5 mg/kg per h) or placebo for 23 h in a double-blind, randomized, crossover study. Venous volume, the diameter of the radial and temporal arteries, calf blood flow and subcutaneous blood flow were measured at baseline and repeated after 1 and 23 h of infusion. RESULTS: Prolonged coadministration of N-acetylcysteine and nitroglycerin potentiated the acute venodilator effect of nitroglycerin as estimated by changes in venous volume (nitroglycerin plus N-acetylcysteine, 4.45 +/- 0.36 ml/100 g; nitroglycerin plus placebo, 3.65 +/- 0.46 ml/100 g, mean +/- SEM, p < 0.05) and prevented development of tolerance as seen after 23 h of treatment with nitroglycerin plus placebo (4.35 +/- 0.25 vs. 3.47 +/- 0.41 ml/100 g, p < 0.05). N-acetylcysteine had no effect on nitroglycerin-induced changes in arterial diameters (p > 0.05) but significantly increased microcirculatory subcutaneous blood flow after 1 h (nitroglycerin plus N-acetylcysteine: 6.3 +/- 1.3 ml/100 g per min vs. nitroglycerin plus placebo: 3.5 +/- 0.3 ml/100 g per min, p < 0.05) and after 23 h (4.4 +/- 0.6 vs. 3.1 +/- 0.5 ml/100 g per min, p < 0.05). CONCLUSIONS: The results suggest that coadministration of nitroglycerin and N-acetylcysteine in humans 1) potentiates and preserves nitroglycerin-induced venodilation and 2) augments the effect of nitroglycerin on small resistance vessels (regulating subcutaneous blood flow) without affecting the response to nitroglycerin in middle-sized arteries. Both the development of nitrate tolerance and the administration of N-acetylcysteine significantly change the normal vasodilator profile of nitroglycerin in humans.

Predictors of coronary in-stent restenosis: importance of angiotensin-converting enzyme gene polymorphism and treatment with angiotensin-converting enzyme inhibitors.

OBJECTIVES: This study aimed to clarify the role of the angiotensin-converting enzyme (ACE) gene polymorphism in the development of in-stent restenosis. BACKGROUND: In-stent restenosis occurs after treatment of coronary artery stenosis in 12% to 32% of coronary interventions with stents. Experimental and clinical studies have suggested that the deletion/insertion (D/I) polymorphism of the ACE gene plays a role in this. METHODS: Quantitative coronary angiography before, immediately after and six months after stent implantation were compared in 369 patients, in whom D/I typing of the ACE gene was performed. RESULTS: At follow-up we found no differences between the three genotypes in minimal lumen diameter (homozygotes with two deletion alleles in the ACE gene [DD], 2.20 mm; heterozygotes with one deletion and one insertion allele in the ACE gene [DI], 2.19 mm; and homozygotes with two insertion alleles in the ACE gene [II], 2.25 mm). The corresponding diameter stenoses were: DD: 25%, DI: 27%, II: 27% (p = NS), and the frequency of restenosis (>50% diameter stenosis) was: DD: 15.7%, DI: 11.0% and II: 16.4% (p = NS). Logistic regression analysis identified diabetes (odds ratio [OR]: 3.0, 95% confidence interval [CI]: 1.0 to 8.7), lesion length (OR: 1.1, 95% CI: 1.01 to 1.30) and minimal lumen diameter immediately after the intervention (OR: 0.3, 95% CI: 0.14 to 0.85) as predictors of in-stent restenosis. In a post hoc analysis of patients treated versus those not treated with an ACE-inhibitor antagonist or an angiotensin receptor antagonist, we found an increased frequency of in-stent restenosis in the DD genotypes (40% vs. 12%, p = 0.006). CONCLUSIONS: The D/I polymorphism is not an independent predictor of coronary in-stent restenosis in general, but it may be of clinical importance in patients treated with ACE inhibitors or angiotensin receptor antagonists.

Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexes with the GluR2 ligand-binding core.

Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid (Br-HIBO), as well as of a mutant thereof (S1S2J-Y702F) in complex with ACPA and Br-HIBO, have been determined. The structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined with the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists.

Increased cardiac BNP expression associated with myocardial ischemia.

Congestive heart failure is accompanied by increased cardiac brain natriuretic peptide (BNP) gene expression with elevated plasma concentrations of BNP and its precursor, proBNP. We investigated if myocardial ischemia in the absence of overt heart failure may be another mechanism for increased myocardial BNP expression. The BNP expression was examined in hypoxic myocardium of patients undergoing coronary bypass grafting surgery, in patients with coronary artery disease and normal left ventricular function undergoing percutaneous transluminal intervention therapy, and in heart failure patients without coronary artery disease. BNP mRNA was quantified by real-time PCR, and plasma BNP and proBNP concentrations were measured with radioimmunoassays. Quantitative analysis of BNP mRNA in atrial and ventricular biopsies from coronary bypass grafting patients revealed close associations of plasma BNP and proBNP concentrations to ventricular, but not atrial, BNP mRNA levels. Plasma BNP and proBNP concentrations were markedly increased in patients with coronary artery disease but without concomitant left ventricular dysfunction. These results are compatible with the notion that myocardial ischemia, even in the absence of left ventricular dysfunction, augments cardiac BNP gene expression and increases plasma BNP and proBNP concentrations. Thus, elevated BNP and proBNP concentrations do not necessarily reflect heart failure but may also result from cardiac ischemia.

Risk factors for thromboembolic complications in chronic atrial fibrillation. The Copenhagen AFASAK study.

As previously reported, 1007 patients with chronic atrial fibrillation participated in the Copenhagen AFASAK study. Before inclusion to trial, they all had a physical examination, chest roentgenogram, and echocardiogram with determination of left atrial size. This study evaluated the importance of cardiovascular risk factors for development of thromboembolic complications. To exclude any treatment effects on occurrence of thromboembolic complications, we included only the 336 patients from the placebo group. Using Cox's regression model, previous myocardial infarction was a significant risk factor for development of thromboembolic complications. Age, gender, heart failure, chest pain, hypertensive heart disease, diabetes, systolic and diastolic blood pressure, smoking, relative heart volume, and left atrial size were all without statistical importance.

Beneficial impact of ramipril on left ventricular hypertrophy in normotensive nonalbuminuric NIDDM patients.

OBJECTIVE: To evaluate the effect of the ACE inhibitor ramipril as compared with placebo on left ventricular mass index (LVMI) in normotensive, nonalbuminuric NIDDM patients with left ventricular hypertrophy (LVH). Patients with NIDDM are characterized by excessive cardiovascular morbidity and mortality, and LVH, an independent risk factor for cardiac events, is often present in NIDDM patients. RESEARCH DESIGN AND METHODS: A total of 38 normotensive, nonalbuminuric (albuminuria < 100 mg/24 h) NIDDM patients with LVH (LVMI > 131 g/m2 in men and > 100 g/m2 in women) were enrolled in a 6-month randomized, double-blind parallel group study to compare the effects of ramipril (5 mg/day) with placebo on LVMI (echocardiography, Vingmed CFM725, Diasonics Sonotron), QTc dispersion determined as the interlead variation in QTc interval on standard electrocardiogram (ECG), and 24-h ambulatory blood pressure (A&D TM2420, Tokyo, Japan). A total of 16 ramipril (10 men, 60 +/- 9 years [mean +/- SD]) and 15 placebo-treated (8 men, 55 +/- 10 years) patients completed the study, and their data are presented. RESULTS: Ambulatory blood pressure was almost identical at baseline (132/76 +/- 3/1 vs. 133/74 +/- 5/2 mmHg [mean +/- SEM]) and remained stable during follow-up (134/76 +/- 3/1 vs. 136/74 +/- 6/2 mmHg) in the ramipril and placebo group, respectively. LVMI was comparable at baseline (137.1 +/- 7.0 vs. 129.6 +/- 3.7 g/m2) in the ramipril and placebo group, respectively, and decreased significantly more in the ramipril group as compared with the placebo group (17.6 +/- 3.0 vs. 5.7 +/- 4.6 g/m2, respectively, 11.9 [0.7-23.1] g/m2, mean difference [95% CI]; P = 0.037). QTc dispersion was comparable at baseline (60.2 [5.5] vs. 64.1 [6.5] ms) and did not change significantly during follow-up: -2.5 [7.0] vs. -12.2 [9.5] ms; mean difference 9.8 (-14.2 to 33.8 ms) in the ramipril and placebo group, respectively. CONCLUSIONS: Ramipril induces regression of LVH in normotensive, nonalbuminuric NIDDM patients, independent of reduction in systemic blood pressure.

Orthotopic cardiac transplantation reverses abnormal reflex regulation of the microvasculature in the lower leg.

OBJECTIVE: The aim was to investigate the effect of cardiac transplantation on reflex control of lower leg subcutaneous blood flow. METHODS: The reflex regulation of subcutaneous blood flow of the lower leg was studied in 11 patients following orthotopic cardiac transplantation, in 11 patients with severe congestive heart failure (New York Heart Association functional class III or IV), and in 11 healthy subjects. Four patients were studied before and after cardiac transplantation. Cause of heart failure was classified as idiopathic dilated cardiomyopathy in all heart failure patients and in all the cardiac transplant patients before transplantation. Blood flow was measured by the local 133xenon washout method in the supine position and during 45 degrees head up tilt. RESULTS: When performing head up tilt without activation of the local nervous venoarteriolar axon reflex in patients with congestive heart failure, the relative subcutaneous blood flow increased abnormally, by 50(SD 25)%, but in patients after cardiac transplantation a normal decrease was seen [-28(13)%, p < 0.001]. The responses in the transplant group were similar to those observed in normal controls with a decrease in blood flow [-32(15)%; NS]. Head up tilt with simultaneous activation of the local venoarteriolar axon reflex increased blood flow [31(22)%] in patients with heart failure as compared to the decrease in blood flow found in the transplants [-44(17)%, p < 0.001]. The decrease of blood flow was not significantly different between the transplant group and control subjects [-53(19)%; NS]. CONCLUSIONS: These results indicate that abnormal reflex regulation in severe congestive heart failure with peripheral vasodilation of the lower leg during orthostasis is reversed and even normalised after cardiac transplantation. The haemodynamic consequence may be a regaining of an oedema-protective mechanism that eliminates the stress (capillary hypertension) on the microcirculation seen in severe heart failure.

Plasma endothelin in congestive heart failure: effect of the ACE inhibitor, fosinopril.

OBJECTIVES: The study evaluates the influence of treatment with the angiotensin-converting enzyme inhibitor, fosinopril, on the plasma endothelin level in patients with congestive heart failure, and the relationship between plasma endothelin and clinical study parameters (bicycle exercise test, echocardiography, heart failure score and blood pressure). METHODS: Plasma endothelin was measured in 34 patients with moderately severe congestive heart failure at randomisation in the fosinopril/placebo-controlled study 'Fosinopril Efficacy and Safety Trial' and at the end of the 12-week study period. RESULTS: The patients had elevated pre-treatment plasma endothelin concentrations (3.5 +/- 1.2 pg/ml, mean +/- s.d., n = 34) compared with healthy volunteers (2.0 +/- 0.4 pg/ml, n = 21, P < 0.0001). Treatment with fosinopril for 12 weeks lowered plasma endothelin from 3.5 +/- 1.2 to 2.5 +/- 0.7 pg/ml (m = 18, P < 0.005), in contrast to the non-significant increase in the placebo-treated group 3.5 +/- 1.3 to 4.3 +/- 2.4 pg/ml, n = 16). A multiple regression analysis for baseline study parameters, demonstrated a significant relationship between plasma endothelin and exercise test duration and a composite heart failure score classification (r = 0.53, P < 0.001). CONCLUSIONS: Treatment of patients with congestive heart failure with the angiotensin-converting enzyme inhibitor, fosinopril, reduce the elevated plasma endothelin level to normal values. The relation between plasma endothelin and clinical parameters indicates that endothelin may play a pathophysiological role in the progression of congestive heart failure.

A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures.

Positive allosteric modulators (PAMs) of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors receive increasing interest as therapeutic drugs and have long served as important experimental tools in the study of the molecular mechanisms underlying glutamate-mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two-electrode voltage-clamp. The dose-response analyses revealed dual effects of NS1376. The modulator induced 30-fold and 42-fold reductions in glutamate potency and increased the glutamate efficacy by 3.2-fold and 5.3-fold at GluA4flip and GluA4flop, respectively. Rapid application of glutamate to excised outside-out patches showed that NS1376 markedly attenuated desensitization, supporting the increased efficacy observed in the oocytes. Furthermore, when applied to acutely isolated mouse brain slices, NS1376 reduced the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus to 51.6 ± 4.3% of baseline, likely as a consequence of reduced glutamate potency. However, the modulator displayed no effects on a sub-maximal long-term potentiation (LTP) protocol. We confirmed that CTZ increases presynaptic transmitter release, a property which was not shared by NS1376. Finally, we obtained detailed molecular information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing agonist efficacy while markedly reducing agonist potency. These unique properties add new aspects to the complexity of allosteric modulations in neuronal systems.

Structure and function of the N-linked glycans of HBP/CAP37/azurocidin: crystal structure determination and biological characterization of nonglycosylated HBP.

The three N-glycosylation sites of human heparin binding protein (HBP) have been mutated to produce a nonglycosylated HBP (ng-HBP) mutant. ng-HBP has been crystallized and tested for biological activity. Complete X-ray data have been collected to 2.1 A resolution, and the structure has been fully refined to an R-factor of 18.4% (R(free) 27.7%). The ng-HBP structure reveals that neither the secondary nor tertiary structure have changed due to the removal of the glycosylation, as compared to the previously determined glycosylated HBP structure. Although the primary events in N-linked glycosylation occurs concomitant with polypeptide synthesis and therefore possesses the ability to influence early events in protein folding, we see no evidence of glycosylation influencing the structure of the protein. The root-mean-square deviation between the superimposed structures was 0.24 A (on C alpha atoms), and only minor local structural differences are observed. Also, the overall stability of the protein seems to be unaffected by glycosylation, as judged by the B-factors derived from the two X-ray structures. The flexibility of a glycan site may be determined by the local polypeptide sequence and structure rather than the glycan itself. The biological in vitro activity assay data show that ng-HBP, contrary to glycosylated HBP, mediates only a very limited stimulation of the lipopolysaccharide induced cytokine release from human monocytes. In animal models of fecal peritonitis, glycosylated HBP treatment rescues mice from and an otherwise lethal injury. It appears that ng-HBP have significant effect on survival, and it can be concluded that ng-HBP can stimulate the host defence machinery albeit to a lesser extent than glycosylated HBP.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

Cerebral blood flow before and after cardioversion of atrial fibrillation.

In nine patients with atrial fibrillation (AF) of less than 3 months' duration, CBF was measured the day before and after and again 30 days after electrical cardioversion therapy to sinus rhythm. The day before cardioversion therapy, median CBF (expressed as initial slope index 1, ml/100 g.min-1) was 35.8 and the day after it was 37.1. After 30 days in sinus rhythm, CBF was 39.4 (NS), although the end-tidal PCO2 values were lower than the pretreatment values. After correction for changes in end-tidal PCO2, the median CBF had increased significantly from 35.8 to 40.3 on day 1 and to 46.7 on day 30. The reduced CBF during AF could be a contributing factor in the development of cerebrovascular complications in patients with AF.

Crystallization and preliminary X-ray analysis of a PNA-DNA complex.

Peptide nucleic acids (PNAs) are DNA mimics with a peptide backbone. PNAs are being intensely investigated owing to a potential as gene-targeted drugs. A PNA (H-GTAGATCACT-NH2)-DNA (5'-AGTGATCTAC-3') complex has been crystallized in a tetragonal space group P4(1)22 with cell dimensions a = b = 79.8, c = 99.9 angstrum. The crystals diffract to about 5 angstrum resolution.

GluR2 ligand-binding core complexes: importance of the isoxazolol moiety and 5-substituent for the binding mode of AMPA-type agonists.

X-ray structures of the GluR2 ligand-binding core in complex with (S)-Des-Me-AMPA and in the presence and absence of zinc ions have been determined. (S)-Des-Me-AMPA, which is devoid of a substituent in the 5-position of the isoxazolol ring, only has limited interactions with the partly hydrophobic pocket of the ligand-binding site, and adopts an AMPA-like binding mode. The structures, in comparison with other agonist complex structures, disclose the relative importance of the isoxazolol ring and of the substituent in the 5-position for the mode of binding. A relationship appears to exist between the extent of interaction of the ligand with the hydrophobic pocket and the affinity of the ligand.