A Triplet Label Extends Two-Dimensional Infrared Spectroscopy from Pico- to Microseconds.

In conventional two-dimensional infrared (2D-IR) spectroscopy, the inherently short vibrational lifetimes limit the time window to observe molecular dynamics typically to tens of picoseconds. The rather complex dynamics of organized molecular systems (e.g., glass formers, polymers, membranes, proteins), however, span a wide range of timescales from femto- to microseconds and beyond. Vibrationally Promoted Electronic Resonance (VIPER) 2D-IR negates the limitations of 2D-IR spectroscopy, for its signal decays with the electronic lifetime. Here, we present 2-Isopropylthioxanthone as the first VIPER 2D-IR probe to exploit intersystem crossing, thereby covering even the microsecond timescale. We achieved the required signal-to-noise ratio and resolution by introducing the Fourier-transform approach to the VIPER 2D-IR pulse sequence. Now, we are in a position to monitor dynamics via spectral diffusion several orders of magnitude beyond the vibrational lifetime of 2D-IR labels.

Proton-Coupled Electron Transport in Two Distinct CYBASC Paralogs of Arabidopsis thaliana: A Comparative Characterization of Highly Conserved Tyrosine and Lysine Residues.

CYBASC proteins are ascorbate (AscH-) reducible, diheme b-containing integral membrane cytochrome b561 proteins (cytb561), which are proposed to be involved in AscH- recycling and facilitation of iron absorption. Two distinct CYBASC paralogs from the plant Arabidopsis thaliana, Atcytb561-A (A-paralog) and Atcytb561-B (B-paralog), have been found to differ in their visible-spectral characteristics and their interaction with AscH- and ferric iron chelates. A previously determined crystal structure of the B-paralog provides the first insights into the structural organization of a CYBASC member and implies hydrogen bonding between the substrate AscH- and the conserved lysine residues at positions 77 (B-K77) and 81 (B-K81). The function of the highly conserved tyrosine at position 70 (B-Y70) is not obvious in the crystal structure, but its localization indicates the possible involvement in proton-coupled electron transfer. Here we show that B-Y70 plays a major role in the modulation of the oxidation-reduction midpoint potential of the high-potential heme, EM(bH), as well as in AscH- oxidation. Our results support the involvement of the functionally conserved B-K77 in the stabilization of the dianion Asc2-. These findings are supported by the crystal structure of the B-paralog, but a comparative biochemical and biophysical characterization of the A- and B-paralogs implied distinct and more complex functions of the corresponding residues A-Y69 and A-K76 in the A-paralog. Our results emphasize the need for a high-resolution crystal structure of the A-paralog to illuminate the differences in functional organization between the two paralogs.

Triptans for Acute Migraine Headache: Current Experience With Triptan Use and Prescription Habits in a Tertiary Care Headache Outpatient Clinic: An Observational Study.

BACKGROUND: Triptans are recommended as first-line therapy in the acute phase of a migraine attack. We describe patterns of triptan use in a tertiary care headache outpatient clinic, particularly addressing factors that are associated with triptan discontinuation. METHODS: From December 2009 until August 2012, demographic and clinical data of consecutive patients with migraine were collected. The Headache Impact Test (HIT-6) was used to measure the adverse impact of headaches. Factors associated with triptan discontinuation were analyzed using binary logistic regression. RESULTS: Of 511 migraine patients included, 73.2% (n = 374) were triptan naïve on first consultation. At follow-up, 57.1% of new triptan users (n = 72/126) reported pain-freedom or pain relief after 2 hours, 40.5% (n = 51/126) did not refill their triptan, another 15.1% (n = 19/126) switched to a different type of triptan. Negative response to triptan treatment and substantial or severe headache impact (HIT-6 score ≥56) at first follow-up were associated with triptan discontinuation (OR 0.39 [95% CI 0.20-0.78], P = .007; OR 3.33 [95% CI 1.10-10.03], P = .033). CONCLUSIONS: Although triptans have been in clinical use for more than two decades, many migraine sufferers in our study population were found to be triptan naïve on first consultation. The HIT-6 score may be used as an indicator of triptan adherence and help to identify patients at risk for triptan discontinuation.

Caged CO(2) for the direct observation of CO(2)-consuming reactions.

CO(2)-consuming reactions, in particular carboxylations, play important roles in technical processes and in nature. Their kinetic behavior and the reaction mechanisms of carboxylating enzymes are difficult to study because CO(2) is inconvenient to handle as a gas, exists in equilibrium with bicarbonate in aqueous solution, and typically yields products that show no significant spectroscopic differences from the reactants in the UV/Vis range. Here we demonstrate the utility of 3-nitrophenylacetic acid and related compounds (caged CO(2)) in conjunction with infrared spectroscopy as widely applicable tools for the investigation of such reactions, permitting convenient measurement of the kinetics of CO(2) consumption. The use of isotopically labeled caged CO(2) provides a tool for the assignment of infrared absorption bands, thus aiding insight into reaction intermediates and mechanisms.

The cysteine S-H stretching mode reports on long-range conformational changes in pyruvate oxidase from E. coli.

The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin-dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions-but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein's C-terminal 23 residues (the 'α-peptide'). Here, we relate the redox-induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (-SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys-SH difference signal to Cys88 and Cys494, both being remote from the moving α-peptide and the redox-active flavin cofactor. Yet, when the α-peptide is proteolytically removed, the Cys-SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α-peptide 'transforms' the catalytically complex EcPOX into the catalytically 'simpler' LpPOX.

Brain-derived neurotrophic factor in primary headaches.

Brain derived neurotrophic factor (BDNF) is associated with pain modulation and central sensitization. Recently, a role of BDNF in migraine and cluster headache pathophysiology has been suspected due to its known interaction with calcitonin gene-related peptide. Bi-center prospective study was done enrolling four diagnostic groups: episodic migraine with and without aura, episodic cluster headache, frequent episodic tension-type headache, and healthy individuals. In migraineurs, venous blood samples were collected twice: outside and during migraine attacks prior to pain medication. In cluster headache patients serum samples were collected in and outside cluster bout. Analysis of BDNF was performed using enzyme-linked immunosorbent assay technique. Migraine patients revealed significantly higher BDNF serum levels during migraine attacks (n = 25) compared with headache-free intervals (n = 53, P < 0.01), patients with tension-type headache (n = 6, P < 0.05), and healthy controls (n = 22, P < 0.001). There was no significant difference between patients with migraine with aura compared with those without aura, neither during migraine attacks nor during headache-free periods. Cluster headache patients showed significantly higher BDNF concentrations inside (n = 42) and outside cluster bouts (n = 24) compared with healthy controls (P < 0.01, P < 0.05). BDNF is increased during migraine attacks, and in cluster headache, further supporting the involvement of BDNF in the pathophysiology of these primary headaches.

Mucinous cystadenoma arising in a completely isolated infected ileal duplication cyst.

Gastrointestinal duplications are uncommon congenital lesions that can occur anywhere along the alimentary tract, and the symptoms of which generally develop during infancy or childhood. Completely isolated duplication cysts are an extremely rare variant of duplication, where no communication between the cyst and the adjacent bowel segment is present. We report the unique case of an adult who presented with right lower abdominal pain and systemic signs of inflammation caused by infection of a completely isolated ileal duplication cyst. Histological examination of the cyst additionally revealed a low-grade mucinous cystadenoma. We discuss the clinical presentations, diagnosis and treatment of this rare entity.

Molecular mechanism of uncaging CO2 from nitrophenylacetate provides general guidelines for improved ortho-nitrobenzyl cages.

Femtosecond spectroscopy and quantum chemical calculations provide detailed insights into the specificities of the uncaging mechanism of CO2 from ortho-, meta-, and para-nitrophenylacetate. The emerging general principles allow a rational design of improved ortho-nitrophenyl cages for chemical and biological applications.

Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli.

The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Delta23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Delta23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 A. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P4(3)2(1)2 with two monomers per asymmetric unit, the crystals of Delta23 EcPOX belong to the orthorhombic space group P2(1)2(1)2(1) and contain 12 monomers per asymmetric unit.

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis. Implications for the substrate activation mechanism of this enzyme.

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 A resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106-113 and 292-301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105-113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105-113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.

Phenotypical variability of post-partum reversible cerebral vasoconstriction syndrome.

Reversible cerebral vasoconstriction syndrome is recognized increasingly as a complication of the postpartum period. Our series of four cases illustrates its phenotypical variability, summarizes the diagnostic work-up, and outlines potential treatment strategies for this usually benign but sometimes disabling and life-threatening disease.

New insights into structure-function relationships of oxalyl CoA decarboxylase from Escherichia coli.

The gene yfdU from Escherichia coli encodes a putative oxalyl coenzyme A decarboxylase, a thiamine diphosphate-dependent enzyme that is potentially involved in the degradation of oxalate. The enzyme has been purified to homogeneity. The kinetic constants for conversion of the substrate oxalyl coenzyme A by the enzyme in the absence and presence of the inhibitor coenzyme A, as well as in the absence and presence of the activator adenosine 5'-diphosphate, were determined using a novel continuous optical assay. The effects of these ligands on the solution and crystal structure of the enzyme were studied using small-angle X-ray scattering and X-ray crystal diffraction. Analyses of the obtained crystal structures of the enzyme in complex with the cofactor thiamine diphosphate, the activator adenosine 5'-diphosphate and the inhibitor acetyl coenzyme A, as well as the corresponding solution scattering patterns, allow comparison of the oligomer structures of the enzyme complexes under various experimental conditions, and provide insights into the architecture of substrate and effector binding sites.

Covalently bound substrate at the regulatory site of yeast pyruvate decarboxylases triggers allosteric enzyme activation.

The mechanism by which the enzyme pyruvate decarboxylase from two yeast species is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants, or of enzyme complexes from two yeast species, three of them reported here for the first time, provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighboring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, one of which provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies, providing a consistent picture of the structural changes occurring upon enzyme activation.

[Implementation of thrombolysis in acute stroke--10-year results of the Innsbruck stroke registry]. / Einsatz der Thrombolyse beim akut ischämischen Schlaganfall : 10-Jahres-Ergebnisse an der Stroke Unit Innsbruck.

BACKGROUND: Randomized controlled trials have yielded evidence for the efficacy and safety of intravenous alteplase in the therapy of acute ischemic stroke. A large patient registry has recently confirmed the safe implementation of this therapy in the clinical routine setting. METHODS: Between January 1998 and December 2007 302 stroke patients were treated with 0.9 mg/kg rt-PA at the stroke unit of the Innsbruck University Hospital. Severity and circumstances of the stroke event, indicators of pre- and intrahospital management as well as safety and outcome at three months were prospectively assessed in the local thrombolysis database. RESULTS: The number of patients receiving intravenous thrombolysis increased continuously from 2 patients in 1998 to 67 in 2007 and 87 patients in 2008. 43% of our patients were females. The median age and NIHSS-score on admission was 67 and 16, respectively. The mean onset-to-needle time decreased from 171 min to 110 min--mainly due to a substantial shortening of the door-to-needle time from 105 min to 45 min. A proportion of 41% of our patients were treated in the main working time while 59% received rt-PA during night and weekend service. A total of 38% of our patients were functionally independent at three months (mRS 0-2). Once considering the high initial stroke severity in our patient series and correcting the NIHSS scores to levels usually seen in randomized control trials and patient registries, 56% of our patients would reach a good outcome (mRS 0-2). The rate of symptomatic intracranial bleedings was low at 6.3%. CONCLUSION: Our data reinforce that intravenous thrombolysis is safe in the treatment of acute ischemic stroke in clinical routine setting. Establishment of modern stroke services and the implementation of structural operating procedures have contributed to an increase in the number of treated patients and a parallel decrease in door-to-needle time at our hospital. Widespread educational programs in the general community, introduction of optimized pre-hospital triage algorithms as well as the potential extension of the 3-hour window to 4.5 hours all are suitable measures to further extend the benefit of i.v. thrombolysis to large proportion of stroke patients.

[Juvenile stroke--results from the Austrian Stroke Registry]. / Juveniler Schlaganfall in Osterreich--Daten aus dem Osterreichischen Schlaganfallregister.

In recent years, many aspects of juvenile stroke have been addressed in medium-sized case series. We have analyzed stroke severity, etiology, risk factors, and outcome in different age groups in the large dataset of the Austrian Stroke Unit Registry. In the nationwide Austrian Stroke Unit Registry 13,440 men and women with ischemic stroke or transient ischemic attack were recorded between March 2003 and February 2007. A number of important disease characteristics were documented by a stroke specialist upon admission of a patient and at discharge from the stroke unit and during a 3-month follow-up interview. A total of 749 patients (5.6%) were 18 to 45 years old and 1895 (14.1%) 18 to 55 years. Arterial dissection and cardiac/paradoxical embolism were the most common causes of stroke up to an age of 45. With a steeply increasing frequency of standard vascular risk factors, micro- and macroangiopathic causes of stroke substantially gain weight after the fourth decade of life. At 3-month follow-up, good clinical outcome (mRS 0-2) and death were 88.3% and 1.4% in the young (

Strain and near attack conformers in enzymic thiamin catalysis: X-ray crystallographic snapshots of bacterial transketolase in covalent complex with donor ketoses xylulose 5-phosphate and fructose 6-phosphate, and in noncovalent complex with acceptor aldose ribose 5-phosphate.

Transketolase is a prominent thiamin diphosphate-dependent enzyme in sugar metabolism that catalyzes the reversible transfer of a 2-carbon dihydroxyethyl fragment between a donor ketose and an acceptor aldose. The X-ray structures of transketolase from E. coli in a covalent complex with donor ketoses d-xylulose 5-phosphate (X5P) and d-fructose 6-phosphate (F6P) at 1.47 A and 1.65 A resolution reveal significant strain in the tetrahedral cofactor-sugar adducts with a 25-30 degrees out-of-plane distortion of the C2-Calpha bond connecting the substrates' carbonyl with the C2 of the cofactor's thiazolium part. Both intermediates adopt very similar extended conformations in the active site with a perpendicular orientation of the scissile C2-C3 sugar bond relative to the thiazolium ring. The sugar-derived hydroxyl groups of the intermediates form conserved hydrogen bonds with one Asp side chain, with a cluster of His residues and with the N4' of the aminopyrimidine ring of the cofactor. The phosphate moiety is held in place by electrostatic and hydrogen-bonding interactions with Arg, His, and Ser side chains. With the exception of the thiazolium part of the cofactor, no structural changes are observable during intermediate formation indicating that the active site is poised for catalysis. DFT calculations on both X5P-thiamin and X5P-thiazolium models demonstrate that an out-of-plane distortion of the C2-Calpha bond is energetically more favorable than a coplanar bond. The X-ray structure with the acceptor aldose d-ribose 5-phosphate (R5P) noncovalently bound in the active site suggests that the sugar is present in multiple forms: in a strained ring-closed beta-d-furanose form in C2-exo conformation as well as in an extended acyclic aldehyde form, with the reactive C1 aldo function held close to Calpha of the presumably planar carbanion/enamine intermediate. The latter form of R5P may be viewed as a near attack conformation. The R5P binding site overlaps with those of the leaving group moieties of the covalent donor-cofactor adducts, demonstrating that R5P directly competes with the donor-derived products glyceraldehyde 3-phosphate and erythrose 4-phosphate, which are substrates of the reverse reaction, for the same docking site at the active site and reaction with the DHEThDP enamine.

The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography.

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.

Radical phosphate transfer mechanism for the thiamin diphosphate- and FAD-dependent pyruvate oxidase from Lactobacillus plantarum. Kinetic coupling of intercofactor electron transfer with phosphate transfer to acetyl-thiamin diphosphate via a transient FAD semiquinone/hydroxyethyl-ThDP radical pair.

The thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent pyruvate oxidase from Lactobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Central to the catalytic sequence, two reducing equivalents are transferred from the resonant carbanion/enamine forms of alpha-hydroxyethyl-ThDP to the adjacent flavin cofactor over a distance of approximately 7 A, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1H NMR-based intermediate analysis indicate that both processes are kinetically coupled. In the presence of phosphate, intercofactor electron-transfer (ET) proceeds with an apparent first-order rate constant of 78 s(-1) and is kinetically gated by the preceding formation of the tetrahedral substrate-ThDP adduct 2-lactyl-ThDP and its decarboxylation. No transient flavin radicals are detectable in the reductive half-reaction. In contrast, when phosphate is absent, ET occurs in two discrete steps with apparent rate constants of 81 and 3 s(-1) and transient formation of a flavin semiquinone/hydroxyethyl-ThDP radical pair. Temperature dependence analysis according to the Marcus theory identifies the second step, the slow radical decay to be a true ET reaction. The redox potentials of the FAD(ox)/FAD(sq) (E1 = -37 mV) and FAD(sq)/FAD(red) (E2 = -87 mV) redox couples in the absence and presence of phosphate are identical. Both the Marcus analysis and fluorescence resonance energy-transfer studies using the fluorescent N3'-pyridyl-ThDP indicate the same cofactor distance in the presence or absence of phosphate. We deduce that the exclusive 10(2)-10(3)-fold rate enhancement of the second ET step is rather due to the nucleophilic attack of phosphate on the kinetically stabilized hydroxyethyl-ThDP radical resulting in a low-potential anion radical adduct than phosphate in a docking site being part of a through-bonded ET pathway in a stepwise mechanism of ET and phosphorolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry.

The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A.

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.