X-ray Thomson Scattering in Warm Dense Matter without the Chihara Decomposition.

X-ray Thomson scattering is an important experimental technique used to measure the temperature, ionization state, structure, and density of warm dense matter (WDM). The fundamental property probed in these experiments is the electronic dynamic structure factor. In most models, this is decomposed into three terms [J. Chihara, J. Phys. F 17, 295 (1987)] representing the response of tightly bound, loosely bound, and free electrons. Accompanying this decomposition is the classification of electrons as either bound or free, which is useful for gapped and cold systems but becomes increasingly questionable as temperatures and pressures increase into the WDM regime. In this work we provide unambiguous first principles calculations of the dynamic structure factor of warm dense beryllium, independent of the Chihara form, by treating bound and free states under a single formalism. The computational approach is real-time finite-temperature time-dependent density functional theory (TDDFT) being applied here for the first time to WDM. We compare results from TDDFT to Chihara-based calculations for experimentally relevant conditions in shock-compressed beryllium.

[18 FDG PET imaging of the brain in the analysis of a dementia syndrome]. / De rol van de 18-fDG PET-scan bij de diagnose dementie.

According to current dementia guidelines from 2005 (CBO) functional brain imaging by PET-scan of the brain has no place in the analysis of a dementia syndrome. Differential diagnosis between Alzheimer disease and other causes of dementia remains important because there are differences in natural course and treatment. Here we present three patients aged 62, 71 and 68 years with dementia syndrome who were assessed at an outpatient memory clinic. After geriatric assessment and subsequent brain MRI the etiology of the dementia remained unclear. In all three patients the etiology became clear after using a 18 FDG PET-scan of the brain. We conclude that 18 FDG PET imaging of the brain has added value in the analysis of dementia syndrome with an unclear etiology after initial analysis.

Dependence of Spurious Charge-Transfer Excited States on Orbital Exchange in TDDFT: Large Molecules and Clusters.

Time-dependent density functional theory (TDDFT) is a powerful tool allowing for accurate description of excited states in many nanoscale molecular systems; however, its application to large molecules may be plagued with difficulties that are not immediately obvious from previous experiences of applying TDDFT to small molecules. In TDDFT, the appearance of spurious charge-transfer states below the first optical excited state is shown to have significant effects on the predicted absorption and emission spectra of several donor-acceptor substituted molecules. The same problem affects the predictions of electronic spectra of molecular aggregates formed from weakly interacting chromophores. For selected benchmark cases, we show that today's popular density functionals, such as purely local (Local Density Approximation, LDA) and semilocal (Generalized Gradient Approximation, GGA) models, are qualitatively wrong. Nonlocal hybrid approximations including both semiempirical (B3LYP) and ab initio (PBE1PBE) containing a small fraction (20-25%) of Fock-like orbital exchange are also susceptible to such problems. Functionals that contain a larger fraction (50%) of orbital exchange like the early hybrid (BHandHLYP) are shown to exhibit far fewer spurious charge-transfer (CT) states at the expense of accuracy. Based on the trends observed in this study and our previous experience we formulate several practical approaches to overcome these difficulties providing a reliable description of electronic excitations in nanosystems.

Role of TNF-alpha and its 55 and 75 kDa receptors in bronchial hyperreactivity.

The pathophysiological role of the tumour necrosis factor (TNF) system was studied in adults (n=37) and children (n=43) non asthmatic offspring of asthmatic parents with and without bronchial hyperreactivity proved by methacholine airway challenge test. SerumTNFalpha and its soluble receptors (sTNF-R1 and R2) were determined by enzyme-linked immunosorbent assay (ELISA). Significantly elevated TNFalpha (adults: mean +/- SD=5.18 +/- 0.87 pg ml(-1), children: 5.08 +/- 1.78) vs. non-hyperreactives (adults: 4.12 +/- 0.43, P < 0.0001, children: 3.75 +/- 0.68, P=0.0084), sTNF-R1 (adults: 144 +/- 0.31 ng ml(-1), children: 1.30 +/- 0 25 vs. adults: 1.21 +/- 0.14, P=0.0305, children: 1.13+/-0.11 ng ml(-1), P=0.0042) and sTNF-R2 (adults: 0.85 +/- 0.40ng ml(-1), children: 0.70 +/- 0.46 vs. adults: 0.56 +/- 0.56 P=0.0084, children: 0.33 +/- 0.17, P=0.0048) and decreased sTNF-R1/R2 ratio (adults: mean +/- SD=0.96 +/- 0.73, children: 2.85 +/- 2.06 vs. adults: 4.82+/-3.40, P=0.0272, children: 4 42 +/- 2 30, P=0.0167) were measured in patients with bronchial hyperreactivityThe provocation doses of methacholine causing a 20% reduction (PD20) in forced expiratory volume in 1 sec (FEV1) were found to be in a significant negative linear correlation with TNFalpha sTNF-R1 and R2 levels in hyperreactive adults and with TNFalpha, sTNF-R2 in hyperreactive children. TNFalpha correlated significantly with its receptors both in hyperreactive adults and children and with the body mass index (BMI) values of adults. The TNF system may contribute to the pathophysiology of bronchial hyperreactivity Altered shedding of sTNF-R1 seems to occur in hyperreactive patients.

The role of active site residue arginine 218 in firefly luciferase bioluminescence.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed a working model of the luciferase active site based on the X-ray structure of the enzyme without bound substrates. In our model, the side chain guanidinium group of Arg218 appears to be located in close proximity to the substrate's hydroxyl group at the bottom of the luciferin binding pocket. A similar role for Arg337 also has been proposed. We report here the construction, purification, and characterization of mutant luciferases R218A, R218Q, R218K, R337Q, and R337K. Alteration of the Arg218 side chain produced enzymes with 15-20-fold increases in the Km values for luciferin. The contrasting near-normal Km values for luciferin determined with the Arg337 enzymes support our proposal that Arg218 (and not Arg337) is an essential luciferin binding site residue. Bioluminescence emission studies indicated that in the absence of a positively charged group at position 218, red bioluminescence was produced. Based on this result and those of additional fluorescence experiments, we speculate that Arg218 maintains the polarity and rigidity of the emitter binding site necessary for the normal yellow-green emission of P. pyralis luciferase. The findings reported here are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

The role of lysine 529, a conserved residue of the acyl-adenylate-forming enzyme superfamily, in firefly luciferase.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen in a two-step process. The enzyme first catalyzes the adenylation of the carboxylate substrate luciferin with Mg-ATP followed by the oxidation of the acyl-adenylate to the light-emitting oxyluciferin product. The beetle luciferases are members of a large family of nonbioluminescent proteins that catalyze reactions of ATP with carboxylate substrates to form acyl-adenylates. Formation of the luciferase-luciferyl-AMP complex is a specific example of the chemistry common to this enzyme family. Site-directed mutants at positions Lys529, Thr343, and His245 were studied to determine the effects of the amino acid changes at these positions on the individual luciferase-catalyzed adenylation and oxidation reactions. The results suggest that Lys529 is a critical residue for effective substrate orientation and that it provides favorable polar interactions important for transition state stabilization leading to efficient adenylate production. These findings as well as those with the Thr343 and His245 mutants are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color.

Under physiological conditions firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen. In nature, bioluminescence emission by beetle luciferases is observed in colors ranging from green (approximately 530 nm) to red (approximately 635 nm), yet all known luciferases use the same luciferin substrate. In an earlier report [Branchini, B. R., Magyar, R. M., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319], we described the effects of mutations at His245 on luciferase activity. In the context of molecular modeling results, we proposed that His245 is located at the luciferase active site. We noted too that the H245 mutants displayed red-shifted bioluminescent emission spectra. We report here the construction and purification of additional His245 mutants, as well as mutants at residues Lys529 and Thr343, all of which are stringently conserved in the beetle luciferase sequences. Analysis of specific activity and steady-state kinetic constants suggested that these residues are involved in luciferase catalysis and the productive binding of substrates. Bioluminescence emission spectroscopy studies indicated that point mutations at His245 and Thr343 produced luciferases that emitted light over the color range from green to red. The results of mutational and biochemical studies with luciferase reported here have enabled us to propose speculative mechanisms for color determination in firefly bioluminescence. An essential role for Thr343, the participation of His245 and Arg218, and the involvement of bound AMP are indicated.

Site-directed mutagenesis of histidine 245 in firefly luciferase: a proposed model of the active site.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We previously reported [Branchini, B. R., Magyar, R. A., Marcantonio, K. M., Newberry, K. J., Stroh, J. G., Hinz, L. K., and Murtiashaw, M. H. (1997) J. Biol. Chem. 272, 19359-19364] that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analogue, was a potent photoinactivation reagent for luciferase. We identified a luciferase peptide 244HHGF247, the degradation of which was directly correlated to the photooxidation process. We report here the construction and purification of wild-type and mutant luciferases H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins are consistent with His245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His245 is oxidized to aspartate during the photooxidation reaction was supported by the extremely low specific activity ( approximately 300-fold lower than WT) of the H245D mutant. Using the crystal structures of luciferase without substrates [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298] and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 [Conti, E., Stachelhaus, T., Marahiel, M. A., and Brick, P. (1997) EMBO J. 16, 4174-4183] as a starting point, we have performed molecular-modeling studies and propose here a model for the luciferase active site with substrates luciferin and Mg-ATP bound. We have used this model to provide a structure-based interpretation of the role of 244HHGF247 in firefly bioluminescence.

Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.

Mutation of a protease-sensitive region in firefly luciferase alters light emission properties.

Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in cell biology. One of its distinctive properties is a pronounced susceptibility to proteolytic degradation that causes luciferase to have a very short intracellular half-life. To define the structural basis for this behavior and possibly facilitate the design of more stable forms of luciferase, limited proteolysis studies were undertaken using trypsin and chymotrypsin to identify regions of the protein whose accessible and flexible character rendered them especially sensitive to cleavage. Regions of amino acid sequence 206-220 and 329-341 were found to be sensitive, and because the region around 206-220 had high homology with other luciferases, CoA ligases, and peptidyl synthetases, this region was selected for mutagenesis experiments intended to determine which of its amino acids were essential for activity. Surprisingly, many highly conserved residues including Ser198, Ser201, Thr202, and Gly203 could be mutated with little effect on the luminescent activity of P. pyralis luciferase. One mutation, however, S198T, caused several alterations in enzymatic properties including shifting the pH optimum from 8.1 to 8.7, lowering the Km for Mg-ATP by a factor of 4 and increasing the half-time for light emission decay by a factor of up to 150. While the S198T luciferase was less active than wild type, activity could be restored by the introduction of the additional L194F and N197Y mutations. In addition to indicating the involvement of this region in ATP binding, these results provide a new form of the enzyme that affords a more versatile reporter system.

Inactivation of firefly luciferase with N-(iodoacetyl)-N'- (5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS).

N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS), a fluorescent reagent that selectively modifies cysteine residues, was demonstrated to irreversibly inhibit native Photinus pyralis luciferase purified from firefly lanterns. Complete inactivation of luciferase activity was accompanied by the blockage of all four cysteine thiols and the concomitant incorporation of 4 mol of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) per mole of enzyme. Employing proteolytic digestions of AEDANS-labeled luciferase and reverse-phase-high-performance liquid chromatography (RP-HPLC), seven tagged peptides were isolated. The AEDANS label provided a convenient spectroscopic marker for the identification of the modified peptides. The sequences of the labeled peptides were deduced from electrospray ionization mass spectrometry (ESMS) and N-terminal sequencing. The fluorescent peptides included cysteine residues and spanned sequences composed of amino acids Leu78-Lys85, Thr214-Arg218, Asp224-Arg275, and Gly388-Met396. The luciferin substrate provided substantial protection against luciferase inactivation resulting in a 60-67% decrease in the labeling of all four cysteine thiols. Thus, it does not appear that a specific cysteine mediates the loss of luciferase activity. Additional LC/ESMS studies permitted the identification of 78% of the native luciferase molecule, which, unlike the recombinant protein, was found to contain an acetylated N-terminus. The AEDANS labeling results and the identification of well-defined proteolytic fragments should facilitate future structure-function investigations of the firefly luciferases.

Abdominal duplex ultrasound screening for occult aortic aneurysm during carotid arterial evaluation.

PURPOSE: The purpose of this study was to investigate the use of a modified abdominal ultrasound examination, which was performed at the time of routine diagnostic carotid artery duplex evaluation, to identify patients at risk for aortic aneurysms. METHODS: A prospective study was undertaken that consisted of a limited ultrasound examination of the abdominal aorta on patients referred to our facilities for duplex carotid evaluation. One hundred thirty-one patients were included in the study; 52% were men and 48% were women, who ranged in age between 40 to 93 years. These patients were not known or suspected to have an abdominal aortic aneurysm. RESULTS: Eleven infrarenal aortic aneurysms (8.4%) were detected, four in patients with positive carotid studies (11%) and seven in those with negative studies (7%); size ranged from 3.0 to 7.5 cm. Categoric data analysis did not produce significance, but a stepwise regression and discriminant analysis model revealed a significant relationship between abnormal velocities and occlusive changes in the carotid arteries and the presence of aortic aneurysms. CONCLUSIONS: Hemodynamically significant carotid artery disease, as diagnosed by duplex examination, appears to be a definite marker for an increased incidence of abdominal aortic aneurysm when compared with the general population. When selective abdominal aortic ultrasonography is done in conjunction with the carotid study, the requirements of additional equipment and technologist time are modest, and the potential benefit to the patient may be significant.

Responding maintained under intermittent schedules of electric-shock presentation: "Safety" or schedule effects?

Four experiments were conducted in which lever pressing by squirrel monkeys was maintained under multiple, mixed, or chained schedules of electric-shock presentation. In the first two experiments, a multiple schedule was employed in which a fixed-interval schedule of shock presentation alternated with a signaled two-minute component. Initially, no events were scheduled during the two-minute component (a safety period). In the first experiment, the safety period was "degraded" by introducing and systematically increasing the frequency of periodic shocks presented during that component. In the second experiment, the proportion of overall safe time to unsafe time was decreased by decreasing the value of the fixed-interval schedule while holding constant shock frequency during the two-minute component. In the third experiment, the overall arrangement was changed from a multiple to a mixed schedule in an attempt to determine whether fixed-interval responding would be maintained when a single exteroceptive stimulus was associated with both components. In the fourth experiment, the overall arrangement was changed from a multiple to a chained schedule in an effort to determine whether fixed-interval responding would be maintained when its consequence was presentation of a signaled "unsafe" period. Fixed-interval responding was well maintained under all experimental conditions; the varied relationships obtained lend more support to conceptualizations of shock-maintained behavior as exemplifying schedule-controlled behavior than to suggestions that such behavior may be readily accounted for by "safety theory."

Tolerance to the increased locomotor activity produced by L-5-hydroxytryptophan following peripheral decarboxylase inhibition in mice.

The development of tolerance to hyperactivity produced by L-5-hydroxytryptophan (5-HTP) was studied in mice pretreated with the peripheral decarboxylase inhibitor MK-486. The results of Experiment I indicated that partial tolerance developed to 5-HTP given twice daily (i.p.) at a dose of 400 mg/kg, but not at a dose of 800 mg/kg. Sustained hyperactivity at the greater dose (800 mg/kg) apparently resulted from the induction of seizures and stereotypy rather than increased locomotor activity. When 5-HTP (400 mg/kg) or saline was administered three times daily (Experiments II and III), the locomotor activity of saline control groups did not differ significantly from chronic 5-HTP-treated groups, but both differed significantly from that of acute 5-HTP-treated animals. Cessation of treatments resulted in a recovery of 5-HTP-induced hyperactivity for experimental animals when later retested. These findings suggest that mice develop tolerance to the effects of 5-HTP on locomotor activity and agree with the hypothesis that behavior change is more closely correlated with the rate of change in concentration of neurotransmitters than the absolute concentrations.