814 research outputs found
Transfer ionization and its sensitivity to the ground-state wave function
We present kinematically complete theoretical calculations and experiments
for transfer ionization in HHe collisions at 630 keV/u. Experiment and
theory are compared on the most detailed level of fully differential cross
sections in the momentum space. This allows us to unambiguously identify
contributions from the shake-off and two-step-2 mechanisms of the reaction. It
is shown that the simultaneous electron transfer and ionization is highly
sensitive to the quality of a trial initial-state wave function
Double photo-ionization of He near a polarizable surface
We calculate the differential cross-section of the direct double
photo-ionization of He physisorbed on a polarizable surface. By including the
influence of the surface potential in the correlated two-electron final state
wavefunction, we show that the differential cross-section carries detailed
information on the electronic correlations at the surface. In particular,
photo-emission along opposite directions, which is prohibited in the free
space, is allowed if the surface potential is long-ranged.Comment: To appear in Phys. Rev. B - Rapid Comm. - 4 pages, 2 PostScript
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Vibrationally Resolved Decay Width of Interatomic Coulombic Decay in HeNe
We investigate the ionization of HeNe from below the He 1s3p excitation to
the He ionization threshold. We observe HeNe ions with an enhancement by
more than a factor of 60 when the He side couples resonantly to the radiation
field. These ions are an experimental proof of a two-center resonant
photoionization mechanism predicted by Najjari et al. [Phys. Rev. Lett. 105,
153002 (2010)]. Furthermore, our data provide electronic and vibrational state
resolved decay widths of interatomic Coulombic decay (ICD) in HeNe dimers. We
find that the ICD lifetime strongly increases with increasing vibrational
state.Comment: 7 pages, 5 figure
Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli
AbstractCalcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 ‘apo’, CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, “Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2” [1]
Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2
Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelinsignalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO- 609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ~17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe267 is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the ‘gatekeeper’ amino acid Phe267Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinasecalmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events.
In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in Escherichia coli.</p
Co-Evolution of quasispecies: B-cell mutation rates maximize viral error catastrophes
Co-evolution of two coupled quasispecies is studied, motivated by the
competition between viral evolution and adapting immune response. In this
co-adaptive model, besides the classical error catastrophe for high virus
mutation rates, a second ``adaptation-'' catastrophe occurs, when virus
mutation rates are too small to escape immune attack. Maximizing both regimes
of viral error catastrophes is a possible strategy for an optimal immune
response, reducing the range of allowed viral mutation rates to a minimum. From
this requirement one obtains constraints on B-cell mutation rates and receptor
lengths, yielding an estimate of somatic hypermutation rates in the germinal
center in accordance with observation.Comment: 4 pages RevTeX including 2 figure
Eine verbesserte fluorimetrische Cortisolbestimmung im Serum
Die fluorimetrische Methode zur Bestimmung von Serumcortisol wurde durch blasenfreie Füllung einer Spezialküvette mittels Pumpvorrichtung, durch Benützung eines Spectralfluorimeters, sowie Verlegung des Meßzeitpunktes (80 min) und durch optimale Anregung (464 nm) und Emissionsmessung (522 nm) verbessert. Empfindlichkeit (<1 µg Cortisol/100 ml), Richtigkeit, Genauigkeit, Reproduzierbarkeit von Tag zu Tag (VK=6–7%) und Störfaktoren der Methode werden angegeben.
Mit dieser Methode wurden Normalberciche für die 9 Uhr-Nüchterncortisolwerte und die i.v. ACTH-Belastung ermittelt.
Bei NNR-insuffizienten Patienten (M. Addison; Zustand nach Operation eines Hypophysentumors; total Adrenalektomierte) wurden i.v. ACTH-Belastungen durchgeführt, wobei sich bereits beim 9 Uhr-Nüchterncortisolwert eine diagnostisch gut brauchbare Trennung gegenüber dem Normalbereich ergab. Unter Dexamethasonsubstitution wurden bei NNR-Insuffizienz sehr niedrige Cortisolspiegel gemessen, was für die Spezifität der Methode spricht.
Dic Bestimmung des 24 Std-Rhythmus der Cortisolwerte bei NNR-Insuffizienten zeigte, daß besonders in den frühen Morgenstunden im Vergleich zu Normalpersonen erniedrigte Cortisolspiegel bestehen. Daraus wird ein besserer Verteilungsvorschlag für die Cortisol-substitution abgeleitet.The fluorimetric determination of serum cortisol was improved
1. using a pumpdevice to fill a special microcuvette avoiding the development of small bubbles,
2. using a recording spectrofluorometer with optimal absorption (464 nm) and emission (522 nm), and
3. allowing for maximal fluorescence of cortisol (80 min).
Sensitivity (<1 µg cortisol/100 ml), accuracy, precision and specificity of the method are reported. Normal values of 9.00 a.m. serum cortisol (9.7–32.0 µg/100 ml) and of values before and after ACTH infusion tests were determined.
For adrenal insufficiency (Addisons disease, total adrenalectomy, or after hypophysectomy) the 9.00 a.m. values of serum cortisol were generally satisfactory for diagnosis. In partial adrenal insufficiency ACTH infusion tests had to be performed.
Very low levels of serum cortisol (2–4 µg/100 ml) were obtained, when patients with adrenal insufficiency were substituted with dexamethasone for three days, proving the specificity of the method.
Determination of circadian rhythms of serum cortisol in patients with adrenal insufficiency on cortisol substitutive therapy in divided doses demonstrated cortisol levels far below the normal values during the carly morning hours. This situation should be improved by dividing the cortisol dose as follows: 6 a.m.: 10 mg, 10 a.m.: 5 mg, 2 p.m.: 5 mg and 8 p.m. or later: 10 mg cortisol
Determination of the Carrier-Envelope Phase of Few-Cycle Laser Pulses with Terahertz-Emission Spectroscopy
The availability of few-cycle optical pulses opens a window to physical
phenomena occurring on the attosecond time scale. In order to take full
advantage of such pulses, it is crucial to measure and stabilise their
carrier-envelope (CE) phase, i.e., the phase difference between the carrier
wave and the envelope function. We introduce a novel approach to determine the
CE phase by down-conversion of the laser light to the terahertz (THz) frequency
range via plasma generation in ambient air, an isotropic medium where optical
rectification (down-conversion) in the forward direction is only possible if
the inversion symmetry is broken by electrical or optical means. We show that
few-cycle pulses directly produce a spatial charge asymmetry in the plasma. The
asymmetry, associated with THz emission, depends on the CE phase, which allows
for a determination of the phase by measurement of the amplitude and polarity
of the THz pulse
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