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.

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.

Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives.

The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.

Safety and efficacy of a new regimen of intravenous recombinant tissue-type plasminogen activator potentially suitable for either prehospital or in-hospital administration.

The safety and efficacy of a new regimen of intravenous recombinant tissue-type plasminogen activator (rt-PA) potentially suitable for either pre- or in-hospital administration were assessed in 60 patients with acute myocardial infarction in an open label coronary angiographic study. The regimen consisted of a 20-mg bolus dose followed 30 min later by a delayed infusion of 80 mg over 2 h. This regimen was designed to facilitate prehospital administration of rt-PA. Infarct-related artery patency (Thrombolysis in Myocardial Infarction [TIMI] grade 2 or 3 flow) was observed in 40 of 53 patients at 60 min (75.5%, 95% confidence intervals [CI] 61% to 84%) and in 55 of 60 patients at 90 min (91.7%, 95% CI 80% to 95%) after the rt-PA bolus. By 90 min the majority of patients (55%) exhibited TIMI grade 3 flow; infarct artery patency at 120 min was 84.9%. During hospitalization definite recurrent ischemia occurred in nine patients (15%); nonfatal recurrent infarction was noted in one (1.7%). Four patients (6.7%) experienced major bleeding, including one with intracranial bleeding. There were seven deaths (11.7%). Mortality was significantly influenced by the occurrence of cardiogenic shock, which was present in five patients at the time of enrollment. Blood fibrinogen levels were obtained before and during rt-PA infusion. At baseline and 30 and 150 min after the bolus dose, the mean fibrinogen level (+/- SD) was 284.83 +/- 77.39, 237.96 +/- 76.92 and 192.04 +/- 57.82 mg/dl, respectively. Compared with the baseline value, there was a significant (p less than 0.05) decrease in fibrinogen at both 30 and 150 min.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly).

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.

Structural and functional studies of hemoglobin Wayne: an elongated alpha-chain variant.

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated alpha-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity (P 1/2 = 0.60 and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect (-delta log P 1/2/pH (7 to 8) = 0.34 and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l' = 5.6 X 10(6) M-1 S-1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l' = 2.6 X 10(6) M-1 S-1), whereas Hb Wayne Asp showed heterogeneous binding (l' = 6.1 X 10(6) and 2.6 X 10(6) M-1 S-1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.

Nonenzymatic glycosylation of human hemoglobin at multiple sites.

The most abundant minor hemoglobin component of human hemolysate is Hb A1c, which has glucose bound to the N-terminus of the beta chain by a ketoamine linkage. Hb A1c is formed slowly and continuously throughout the 120 day lifespan of the red cell. It can be synthesized in vitro by incubating purified hemoglobin with 14C-glucose. Other minor components, Hb A1a1 and Hb A1a2 are adducts of sugar phosphates at the N-terminus of the beta chain. Hb A1b contains an unidentified nonphosphorylated sugar at the beta N-terminus. In addition, a significant portion of the major hemoglobin component (Hb Ao) is also glycosylated by a glucose ketoamine linkage at other sites on the molecule, including the N-terminus of the alpha chain and the epsilon-amino group of several lysine residues on both the alpha and the beta chains. The results indicate that the interaction of glucose and hemoglobin is rather nonspecific and suggests that other proteins are modified in a similar fashion.

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.

Status of equine viral arteritis in Kentucky, 1985.

Clinical cases of equine arteritis virus infection have not been diagnosed in Kentucky since 1984, and there has been no indication that any of the horses involved in the 1984 epizootic have since been responsible for spread of the disease to horses in other states or other countries. Cases of abortion caused by naturally acquired infection with this virus have not been confirmed in 1984 or 1985. Neither field nor vaccine strains of equine arteritis virus have been shown to induce teratologic abnormalities or the carrier state in foals born to infected or vaccinated mares. The carrier stallion appears to have played a major epidemiologic role in the dissemination and perpetuation of the virus. A commercial modified live equine viral arteritis vaccine was found to be safe and efficacious for stallions and mares. The disease can be controlled by immunizing the stallion population and by restricting the breeding of equine arteritis virus-shedding stallions to vaccinated or seropositive mares, followed by an appropriate period of isolation from other nonvaccinated Equidae.

Acute selenium poisoning: case report.

A case of self-poisoning with sodium selenate sheep drench, along with blood and urine levels of selenium, is reported. Treatment included gastric lavage, diuresis, vitamin C, and dimercaprol, and the patient recovered without sequelae.

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.

Assembly of human adult and sickle hemoglobins from their oxygenated subunits. Differential rates of beta chain tetramer dissociation.

The rate of assembly of oxyhemoglobins A and S at pH 7 and 20 degrees C has been followed spectrophotometrically at 582.5 nm, a maximum of the difference spectrum of isolated oxygenated subunits and intact oxyhemoglobin. In 0.1 M potassium phosphate buffer, the resultant time courses following mixing of equivalent concentrations of alpha and beta chains were homogeneous and followed first order kinetics. These time courses were protein concentration independent over a range of 8.0 to 60 X 10(-6) M in heme after mixing and presumably reflect the rate of dissociation of the beta chain tetramer. This rate-limiting dissociation reaction was found to be nearly 2-fold slower for the beta S than the beta A chain tetramer exhibiting half-times of 27 and 15 min, respectively. The overall rate of formation of Hb S and Hb A appears to be significantly influenced by the rates of dissociation of their respective beta chains. These findings are in contrast to mixing experiments done in 0.01 M potassium phosphate buffer which revealed time courses which were heterogeneous and markedly dependent upon protein concentration. The stability of the oxygenated beta chain tetramer and, therefore, the overall kinetic profile of liganded hemoglobin reconstitution is acutely sensitive to buffer conditions.

An investigation of human oxyhemoglobin beta tetramer dissociation using haptoglobin binding.

Haptoglobin was used as a macromolecular probe to investigate the formation of human oxyhemoglobin beta chain dimers from tetramers in 0.1 M potassium phosphate buffer, 20 degrees C at pH 7 and pH 8. Monitoring of spectral changes upon mixing haptoglobin with beta heme chains (2.5 and 5 micromolar) revealed an overall decrease in absorbance accompanied by a shift of the Soret spectral peak from 415 to 417 nm. The magnitude of the absorbance decrease was proportional to the beta concentration; the time courses consistently yielded greater color at pH 8 than at pH 7. At pH 8, two exponential phases of 0.47 min-1 and 0.084 min-1 were seen whose rates remained invariant with concentration. In contrast, only one exponential process was evident at pH 7, yielding a first order rate constant of 0.21 min-1. We have spectrophotometrically followed the beta chain tetramer to dimer dissociation reaction, thus providing information about the contribution of this step to hemoglobin assembly.