Patients' experiences of a diagnosis of Hughes' syndrome.

The objectives of the study were to describe the experience of patients immediately prior to a diagnosis of Hughes syndrome (HS) or antiphospholipid syndrome and post-diagnosis. A questionnaire survey was carried out set in the Hughes Syndrome Foundation, St. Thomas' Hospital, London, 2006. Participants were all patients who are members of the Hughes Syndrome Foundation. The main outcome measures were responses to a questionnaire relating to the experiences of people with a diagnosis of HS, such as number of hospitalisations, number of consultants seen, number of miscarriages, etc. A total of 157 patients completed the questionnaire, giving a response rate of 60.4%. Most (85%) were women and mean age was 46 years (SD 12). The median time to diagnosis was 3 years. The median number of consultants seen was 2 (max 19) with a median time in hospital pre-diagnosis of 10 days. The most common initial diagnoses were migraines, multiple sclerosis and systemic lupus erythematosus. Among women, 46% had had a miscarriage. Two thirds of respondents thought a blood test would have led to an earlier diagnosis. Comments from patients indicated a lack of awareness among specialists and general practitioners. The survey demonstrated a long time lag for diagnosis of Hughes syndrome, with increased costs to the NHS and emotional and financial cost to the patient. Greater awareness of this condition would benefit patients and the NHS.

Microarray analysis and organization of circadian gene expression in Drosophila.

We have used high-density oligonucleotide arrays to study global circadian gene expression in Drosophila melanogaster. Coupled with an analysis of clock mutant (Clk) flies, a cell line designed to identify direct targets of the CLOCK (CLK) transcription factor and differential display, we uncovered several striking features of circadian gene networks. These include the identification of 134 cycling genes, which contribute to a wide range of diverse processes. Many of these clock or clock-regulated genes are located in gene clusters, which appear subject to transcriptional coregulation. All oscillating gene expression is under clk control, indicating that Drosophila has no clk-independent circadian systems. An even larger number of genes is affected in Clk flies, suggesting that clk affects other genetic networks. As we identified a small number of direct target genes, the data suggest that most of the circadian gene network is indirectly regulated by clk.

Consequence of beta 16 and beta 112 replacements on the kinetics of hemoglobin assembly.

The rates of alpha/beta monomer combination of four beta(A) variants (beta 112C --> S, beta 112C --> D, beta 112C --> T, and beta 112C --> V) in the presence and absence of beta 16G --> D (beta(J)) were measured in an attempt to assess the consequences of amino acid substitution at both a surface (beta 16) and an alpha(1)beta(1) interface (beta 112) residue on oxyhemoglobin assembly. Rates of alpha/beta monomer combination determined spectrally in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM EDTA, pH 7.4, at 21.5 degrees C differed by over 40-fold (22 +/- 2.0 to 0.49 +/- 0.1 x 10(5) M(-1) s(-1)), and were in the order: HbA beta 112S = HbJ beta 16D, beta 112S > HbA beta 112D = HbJ beta 16D, beta 112D > HbA > Hb J > HbA beta 112T = HbJ beta 16D, beta 112T > HbJ beta 16D, beta 112V > HbA beta 112V. This extensive kinetic investigation of single/double amino acid-substituted recombinant hemoglobin molecules, in conjunction with molecular modeling studies, has allowed examination of an array of unique alpha/beta subunit interactions and assembly processes.

Wild-type circadian rhythmicity is dependent on closely spaced E boxes in the Drosophila timeless promoter.

Transcriptional regulation plays an important role in Drosophila melanogaster circadian rhythms. The period promoter has been well studied, but the timeless promoter has not been analyzed in detail. Mutagenesis of the canonical E box in the timeless promoter reduces but does not eliminate timeless mRNA cycling or locomotor activity rhythms. This is because there are at least two other cis-acting elements close to the canonical E box, which can also be transactivated by the circadian transcription factor dCLOCK. These E-box-like sequences cooperate with the canonical E-box element to promote high-amplitude transcription, which is necessary for wild-type rhythmicity.

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins.

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of beta heme subunits affected the assembly properties of both Des beta(A) and Des beta(S) with alpha heme chains, albeit to differing degrees. Indeed, the rate of Des beta(A) oligomer dissociation (k1), as determined by visible spectroscopy, was 4.3-fold faster than that of its native beta(A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k'2) from their alpha and beta [Des beta(A)] heme-containing monomers. Matching kinetic analysis of Des beta(A) and Des beta(S) chain assembly (with an identical a chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for beta(A) and beta(S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the beta-6 (A3) residue, and its amino-terminal region, in a chain partner recognition and subsequent human hemoglobin assembly.

Incidence and predictors of myocardial infarction among patients with atrial fibrillation.

OBJECTIVES: We sought to evaluate the utility of excluding myocardial infarction (MI) in patients presenting to the emergency department (ED) with atrial fibrillation (AF) and to identify predictors of MI in this group. BACKGROUND: Patients with AF are frequently admitted to the hospital, in part, to exclude an associated MI. There are no prospective data on unselected patients to support this common practice. METHODS: We conducted a prospective cohort study of all patients who presented to a single-center ED with the primary diagnosis of AF. RESULTS: Of a total of 255 patients, 190 (75%) were admitted to the hospital, and 109 of them underwent a standard "rule-out MI" protocol. Of these 109 patients, six (5.5%) were identified as having an acute MI at the time of admission. Chest pain was present in 39% of patients, with a sensitivity and specificity for the occurrence of MI of 100% and 65%, respectively. ST segment elevation or depression was present in 43% of patients, with a sensitivity and specificity of 100% and 51%. The presence of either major ST segment depression (>2 mm) or elevation on the admission electrocardiogram (ECG) was present in 6%, with a sensitivity of 100% and a specificity of 99%. The resulting positive and negative predictive values were 86% (95% confidence interval [CI] 42% to 99%) and 100% (95% CI 96% to 100%), respectively. Use of this criterion would have reduced the number of rule-out MIs in our study group by 94%, with no loss of sensitivity. CONCLUSIONS: Chest pain and ST segment depression are extremely common findings in patients presenting to the ED with AF and have limited power to predict MI. In contrast, ECG evidence of ST segment elevation or depression >2 mm appears to be a reliable discriminator of which patients are at risk for MI. Patients without significant ST segment changes are at very low risk for MI and may not require performance of the rule-out MI protocol or hospital admission if clinically stable.

takeout, a novel Drosophila gene under circadian clock transcriptional regulation.

We report the identification and characterization of a new Drosophila clock-regulated gene, takeout (to). to is a member of a novel gene family and is implicated in circadian control of feeding behavior. Its gene expression is down-regulated in all of the clock mutants tested. In wild-type flies, to mRNA exhibits daily cycling expression but with a novel phase, delayed relative to those of the better-characterized clock mRNAs, period and timeless. The E-box-containing sequence in the to promoter shows impressive similarities with those of period and timeless. However, our results suggest that the E box is not involved in the amplitude and phase of the transcriptional cycling of to. The circadian delayed transcriptional phase is therefore most likely the result of indirect regulation through unknown transcription factors.

Soret spectroscopic and molecular graphic analysis of human semi-beta-hemoglobin formation.

The interaction of heme-free alpha (alpha(o)) and heme-containing beta (beta(h)) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at 5 degrees C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10(7) M(-1) s(-1). This was 100-fold more rapid than the reported rate of combination of alpha(h) and beta(h) proteins. This encounter-like rate of semi-beta-hemoglobin (alpha(o)beta(h)) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB-GH helical entities. Molecular graphic analysis of modeled alpha(o) protein superimposed over native alpha(h) protein revealed an apo Mb-like structure with well-defined AB-GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the alpha1beta1 interface, was apparently responsible for the enhanced association properties of the alpha(o) protein.

Surface and interface beta-chain residues synergistically affect hemoglobin assembly.

Homo- and heterotetramer formations of beta112 variants (beta(112Cys-->Asp), beta(112Cys-->Ser), beta(112Cys-->Thr), and beta(112Cys-->Val)) of hemoglobin were characterized in the presence and absence of beta(16Gly-->Asp) in vitro. In all cases an alteration in overall surface charge (beta(16Gly-->Asp)) decreased the beta(4) homotetramer stability (association constants as determined by gel-permeation chromatography) albeit to differing extents. In contrast, competition experiments of hemoglobin subunits showed that heterotetramer formation was promoted by this substitution. Order of increase in tetramer formation by the additional negative surface charge in the beta112 variants was as follows: Hb betaG16D, C112D > Hb betaG16D, C112S > Hb betaG16D > Hb G16D, C112T > Hb betaG16D, C112V. Thus, the overall surface charge of the beta chain and its contribution to electrostatic interaction in these instances appear to act in synergy with alpha(1)beta(1) interface residues to affect the assembly of hemoglobin molecules.

Wavelength-dependent spectral changes accompany CN-hemin binding to human apohemoglobin.

The interaction of apohemoglobin with two heme derivatives, CN-protohemin and CN-deutero-hemin, was monitored at multiple Soret wavelengths (417-423 and 406-412 nm, respectively) in 0.05 M potassium phosphate buffer, pH 7.0, at 10 degrees C and revealed, as previously reported, a multiphasic kinetic reaction. Wavelength-dependent reactions were observed for both CN-protohemin and CN-deuterohemin derivatives with the alpha chain (bathochromic entity) displaying faster (4- to 7-fold) rates throughout the courses of both heme-binding reactions. The basis of this spectrally heterogeneous kinetic phenomenon could be deduced from molecular modeling studies of alpha- and beta-chain structures. Key differences in the number of stabilizing contacts of the two chains with the peripheral alpha propionyl 45(CE3); 58(E7); 61(E10) as well as the beta vinyl 38(C4); 71(E15); 106(G8) groups were found. Furthermore, RMS plots comparing apo- and heme-containing subunits reveal substantial structural disparities in the C-CD-F-FG helical regions of the alphabeta dimer interface.

Analysis of the global architecture of hemoglobin A2 by heme binding studies and molecular modeling.

The kinetics of CNProto- and CNDeutero-hemin binding to apohemoglobin A2 was investigated in a stopped-flow device in 0.05 M potassium phosphate buffer, pH 7, at 10 degrees C. The overall kinetic profile exhibited multiple phases: Phases I-IV corresponding with heme insertion (8.5-13 x 10(7) M(-1) s(-1)), local structural rearrangement (0.21-0.23 s(-1)), global alphadelta structural event (0.071-0.098 s(-1)), and formation of the Fe-His bond (0.009-0.012 s(-1)), respectively. Kinetic differences observed between apohemoglobin A2 and apohemoglobin A (previously studied) prompted an analysis of the structures of beta and delta chains through molecular modeling. This revealed a structural repositioning of the residues not only at, but also distant from the site of the amino acid substitutions, specifically those involved in the heme contact and subunit interface. A significant global change was observed in the structure of the exon-coded 3 region and provided additional evidence for the designation of this as the subunit assembly domain.

Fluorescence studies of human semi-beta-hemoglobin assembly.

The intrinsic fluorescence properties of human alpha apohemoglobin at protein concentrations from 1 to 5 microM in 0.1 M potassium phosphate buffer, pH 7 or 8 at 5 degrees C were monitored in the absence and presence of a fixed concentration (5 microM) of a fluorescence quenching heme-containing native or Des (146-His, 145-Tyr) beta chain partner. These "reverse quenching" studies revealed that the emission intensity changes observed correlated well with protein concentration and theoretical extent of semi-beta-hemoglobin assembly. Furthermore, the relative quenching efficiencies were calculated to be 0.32, 0.25 and 0.61 for beta (pH 7), beta (pH 8) and Des beta (pH 7) chains, respectively. Thus, heme-mediated quenching was sensitive to the expected pH induced alpha apohemoglobin conformational change and to alteration in beta chain structure. Intramolecular changes induced by carboxylterminal modification (decreased "beta chain self-quenching") appeared to enhance the intermolecular rearrangements (increased "alpha chain partner quenching") seen upon subunit assembly.

Rendu-Osler-Weber syndrome: a current perspective on cerebral manifestations.

Rendu-Osler-Weber syndrome (hereditary haemorrhagic telangiectasia) is a vascular dysplasia characterized by recurrent epistaxis, mucocutaneous telangiectasia and a family history of the disorder. Although rare, it may cause significant morbidity to healthy and young individuals. We report three cases highlighting the cerebral manifestations of this disorder. These cases include cerebral abscess, cerebral haemorrhage and embolic stroke. These cerebral manifestations are due to complications associated with pulmonary arteriovenous fistulae or cerebral vascular malformations. The current management and screening for this disorder are reviewed.

Spectral demonstration of semihemoglobin formation during CN-hemin incorporation into human apohemoglobins.

The incorporation of CN-hemin into three human adult apohemoglobin species (apohemoglobin, alpha-apohemoglobin, and apohemoglobin modified at its beta93 sulfhydryl with p-hydroxymercuribenzoate) has been monitored at micromolar concentrations in 0.05 M potassium phosphate buffer, pH 7.0, at 10 degrees C. In all cases, Soret spectral blue shifts accompanied CN-protohemoglobin but not CN-deuterohemoglobin formation. This finding in conjunction with isofocusing studies provided evidence of a CN-protosemi-alpha-hemoglobin intermediate, the formation of which appeared to be a direct consequence of CN-protohemin-alpha heme pocket interactions. The kinetics of full reconstitution of CN-protohemoglobin and CN-deuterohemoglobin revealed four distinct phases that apparently correlated with heme insertion (Phase I), local structural rearrangement (Phase II), global conformational response (Phase III), and irreversible histidine iron bond formation (Phase IV). These phases exhibited rates of 7.8-22 x 10(7) M(-1) s(-1), 0.19-0.23 s(-1), 0.085-0.12 s(-1), and 0.008-0.012 s(-1), respectively. Partial (50%) reconstitution with CN-protohemin, in contrast, revealed only three kinetic phases (with Phase III missing) of heme incorporation into native and p-hydroxymercuribenzoate-modified apohemoglobin. Furthermore, the absence of Phase III slowed the rate of proximal bond formation. These findings support the premise that irreversible assembly of CN-protosemi-alpha-hemoglobin is deterred by the presence of a heme-free beta partner, the consequence of which may be that intermolecular heme transfer is encouraged under conditions of heme deficiency in vivo.

Carboxyl-terminal modification influences subunit assembly of sickle hemoglobin beta chains.

The subunit assembly properties of isolated beta and Des(His-146,Tyr-145) beta chains of sickle hemoglobin were investigated by isoelectric focusing over a protein concentration range from 500-125 microM in heme. Two components (presumably tetramer and monomer) and three components (designated tetramer, dimer and monomer) were visualized for beta s and Des(His-146,Tyr-145) beta s chains, respectively. Intensitometric quantitation of Des(His-146,Tyr-145) beta s chains demonstrated a similar distribution of all three structural components before and after the addition of their heteropartner alpha chains. This is in direct contrast to the reported preferential loss of Des(His-146,Tyr-145) beta A monomer species upon assembly and points to a major role of the beta 6 residue in the overall structural homeostasis of carboxylterminal modified human beta chains.

Fluorescence studies of normal and sickle beta apohemoglobin self-association.

The acrylamide quenching of the intrinsic tryptophanyl fluorescence of normal and sickle beta apohemoglobins has been studied in 0.05 M potassium phosphate buffer, pH 7.5, at 5 degrees C over a protein concentration range from 1 to 50 microM. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluorescence of both normal and sickle beta apohemoglobins, suggesting that one tryptophanyl residue [presumably that at position 37(C3)], was more accessible to collisional quencher than the other beta tryptophanyl residue [15(A12)]. Additional studies, which altered viscosity and subunit assembly experimental parameters, supported the assignment of residue 37 as the more dynamically accessible residue. Finally, the quenching data were also found to be dependent on protein concentration, implying that this difference in the mobility between the two residues is a sensitive probe of self-aggregation. Extrapolated dynamic quenching constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and sickle beta apohemoglobins, and were found to be 5.6 and 2.4 microM, respectively, thus demonstrating distinct self-association properties of beta A and beta S apohemoglobins.

Effect of carboxyterminal modification on the oligomeric structure of human beta hemoglobin.

A broad beta chain band region containing multiple components was observed with both native beta and Des(His-146, Tyr-145) beta chains following isoelectric focusing on agarose gels (pH 6.0-8.0). In contrast to the tetramer-monomer system of beta chains, a distinct separation of three components (tetramer, dimer and monomer) was seen for Des(His-146, Tyr-145) beta chains indicative of an oligomeric structural beta model with a stable dimer species. Protein dilution (500 to 15.6 microM in heme) amplified the more cathodic (presumably dimeric and monomeric) components of these chains, and titration with partner alpha chains resulted in a selective depletion of the monomer (most cathodic) component which could be quantitatively correlated with assembly of the hemoglobin tetramer.

Monitoring the effect of subunit assembly on the structural flexibility of human alpha apohemoglobin by steady-state fluorescence.

A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14(A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104(G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free human alpha-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer, pH 7.0, at 5 degrees C, and the structural flexibility of alpha-apohemoglobin, in the absence and presence of human beta-heme-containing chains, was examined by a steady-state solute quenching technique. The quenched efficiencies (EQ) and Förster distances (R0Q) were analyzed by least-squares to determine the goodness of fit (chi R2) for the assumed distribution parameters: average distance r and half-width hw. Data for alpha-apohemoglobin in the absence and presence of beta h chains yielded values for r of 18 and 22 A and hw of 20 and 8.5 A, respectively. Although the increase in r for alpha-apohemoglobin in the presence of beta h chains was presumably a consequence of additional quenching from the heme moiety, the change in the half-width strongly indicated a decrease in the flexibility of the alpha-apohemoglobin chain within the assembled protein. A transition in structural flexibility similar to that demonstrated here may be an important aspect of human hemoglobin assembly.

Steady state fluorescence energy transfer measurements of human alpha apohemoglobin structure.

A nonfluorescent reagent, 4-phenylazophenylmaleimide [4-PAPM], was attached to the sole cysteine residue [104(G11)] of alpha apohemoglobin (alpha degree) and served as an energy acceptor for the single intrinsic tryptophanyl [14(A12)] donor. This novel fluorescence system provided a transmolecular vehicle by which the overall structure of alpha degree could be monitored in 0.05 M potassium phosphate buffer at 5(0) C. Ratio of the emission intensities at 335 nm for monomeric solutions (5 x 10(-6) M) of both alpha degree and alpha degree [4-PAPM] furnished a measure of the efficiency of energy transfer and average distance of separation (r). An apparent increase in the value of r was observed from pH 6.5 to 8.5, suggesting that the conformation (the structural relationship of the A and G helical segments) of alpha degree is responsive to its electrostatic environment.

Role of alpha and beta carboxyl-terminal residues in the kinetics of human oxyhemoglobin dimer assembly.

Human oxyhemoglobin assembly was evaluated in the Soret region by rapidly mixing normal and carboxypeptidase-digested chains (1-10 x 10(-6) M, heme basis) in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM EDTA, pH 7.4, at 21.5 degrees C. Rate constants of 1.14 (+/- 0.09) and 2.11 (+/- 0.06) x 10(5) M-1 S-1 were measured for the association of Des(Arg-141) alpha with beta A and alpha A with beta A chains, respectively. The slower combination rate of Des(Arg-141) alpha with beta A chains is in agreement with that predicted solely on the basis of electrostatic considerations, as are the measured rate constants of 0.75 (+/- 0.12) and 1.86 (+/- 0.08) x 10(5) M-1 S-1 obtained for the combination of Des(Arg-141) alpha with variant beta S (Glu-6-->Val) and beta N Baltimore (Lys-95-->Glu) chains, respectively. However, the combination rates of alpha A and Des(Arg-141) alpha with beta A chains measured in the pH range from 7.0 to 9.0 demonstrated that the altered overall surface charge was not the only determinant of the assembly rates observed for Des(Arg-141) alpha chains. Furthermore, a rate constant of 11.3 (+/- 0.05) x 10(5) M-1 S-1 (which is 5.4-fold faster than the rate of alpha A beta A dimer assembly) was observed for Des(His-146,Tyr-145) beta chains. These kinetic studies suggest a critical role for the carboxyl-terminal domain in the assembly of human hemoglobin in vitro and perhaps in vivo.