Investigation of boron carbide and iridium thin films, an enabling technology for future x-ray telescopes.

We present an experimental examination of iridium and boron carbide thin-film coatings for the purpose of fabricating x-ray optics. We use a combination of x-ray reflectometry and x-ray photoelectron spectroscopy to model the structure, composition, density, thickness, and micro-roughness of the thin films. We demonstrate in our analyses how the two characterization techniques are complementary, and from this we derive that an overlayer originating from atmospheric contamination with a thickness between 1.0-1.6 nm is present on the surface. The magnetron sputtered iridium films are measured to have a density of 22.4g/cm3. The boron carbide film exhibits a change in chemical composition in the top ∼2nm of the film surface when exposed to the ambient atmosphere. The chemical reaction occurring on the surface is due to an incorporation of oxygen and hydrogen present in the ambient atmosphere. Lastly, we present a correlation between the absorption edges and the emission lines exhibited by the thin films in an energy range from 50-800 eV and the impact on the reflectivity performance due to contamination in thin films.

Four-dimensional velocity mapping of the hepatic and splanchnic vasculature with radial sampling at 3 tesla: a feasibility study in portal hypertension.

PURPOSE: To demonstrate the feasibility of PC-VIPR (Phase Contrast Vastly undersampled Imaging with Projection Reconstruction) for the depiction and hemodynamic analysis of hepatic and splanchnic vessels in patients with portal hypertension. MATERIALS AND METHODS: Twenty-four cirrhotic patients (55.9 ± 10.4 years) were scanned using 5-point PC-VIPR for high spatial resolution imaging with large volume coverage at 3 Tesla (T) using a 32-channel body coil. Vessel segmentation and hemodynamic visualization included color-coded three-dimensional (3D) streamlines and particle traces. Segmentation quality was compared with contrast-enhanced multi-phase liver imaging. Flow pattern analysis was performed in consensus of three readers. The MELD score was calculated to estimate disease severity and was correlated to image quality. RESULTS: Good to excellent visualization quality was achieved in 23/24 cases. All arterial vessels and 144/168 vessels of the portal venous (PV) circulation were unambiguously identified. No correlation with the MELD score was found. Eight of 148 vessels of the PV circulation demonstrated reverse (hepatofugal) flow. Hepatofugal flow in small tributaries to PV flow were present in three cases despite hepatopetal flow in the PV. CONCLUSION: This feasibility study demonstrates the feasibility of PC-VIPR for simultaneous morphological and hemodynamic assessment of the hepatic and splanchnic vasculature in cirrhosis and portal hypertension. Future studies with quantitative analyses are warranted.

Correlation between the induction of heat shock protein 70 and enhanced viral reactivation in mammalian cells treated with ultraviolet light and heat shock.

Enhanced viral reactivation (EVR) is considered to be one manifestation of an inducible response to DNA damage in mammalian cells analogous to the SOS response in Escherichia coli. EVR is characterized by the increased survival of ultraviolet (UV)-irradiated virus in cells which have been pretreated with DNA-damaging agents or by another type of cellular stress, heat shock (HS). In this study, we have analyzed the induction of nuclear proteins from Vero cells treated with either UV or HS, with the goal of identifying the protein(s) which mediate the EVR response. Results of 2-dimensional protein gel electrophoresis and fluorographic analysis of [35S]methionine-labeled nuclear proteins showed that UV-irradiation caused the increased synthesis of five proteins at 4-9 h after treatment. At 19-24 h, one of these proteins was still being synthesized at a higher level in UV-irradiated cells, and there were nine additional proteins whose syntheses were enhanced over control levels. In contrast, HS induced only one Mr 72,000 nuclear protein whose synthesis was maximal during the 4-9-h labeling period and corresponded to one of the proteins induced by UV at 19-24 h. Subsequent Western and Northern blot analyses have confirmed that this protein is a member of the heat shock protein (hsp) 70 family. Elevated nuclear levels of this protein correlated temporally with the maximum EVR response induced by each treatment (4 h after HS and 24 h after UV). Since the kinetics of EVR is different following UV and HS and parallels the difference in the induction of nuclear levels of hsp70 following each treatment, the results suggest that hsp70 may be involved in mediating the EVR response. In addition, this protein may also play a role in the recovery of DNA synthesis in UV-irradiated cells.

Plasmodium falciparum: susceptibility in vitro and in vivo to chloroquine and sulfadoxine-pyrimethamine in Ghanaian schoolchildren.

In Ghana, resistance of Plasmodium falciparum to chloroquine was observed for the first time in 1986. By the end of 1991 it had reached a high frequency and a substantial degree. A combined study in vivo and in vitro of the response of P. falciparum to chloroquine and sulfadoxine/pyrimethamine was carried out in Madina, Accra, in the coastal area of Ghana, late in 1991. 96 valid tests in vivo were performed with children and adolescents. There were 52 successful tests in vitro with chloroquine, and 52 with sulfadoxine/pyrimethamine. 45% of the chloroquine tests in vivo and 37% of the sulfadoxine/pyrimethamine tests in vivo indicated RII/RIII resistance. Results in vivo and in vitro were significantly correlated. The presence of RIII responses, 9% with chloroquine and 14% with sulfadoxine/pyrimethamine, indicates a need for third-line antimalarial drugs, the unregulated use of which may entail the risk of early and rapid occurrence of multi-resistance.

Chloroquine resistance of Plasmodium falciparum: a biological advantage?

The response in vitro of Plasmodium falciparum to chloroquine, mefloquine and quinine was studied in a hyperendemic peri-urban area of Accra, Ghana, during the fourth quarter of 1991, yielding a total of 159 valid tests. Schizont maturation in drug-free controls and effective chloroquine concentrations were strongly correlated. This was not seen with mefloquine or quinine. Higher mean parasitaemia in untreated oligo-symptomatic carriers of overtly chloroquine-resistant P. falciparum than in carriers of more sensitive parasites was another indication of higher viability and biological advantage of chloroquine-resistant P. falciparum that may conceivably have clinical implications.

Activity of benflumetol and its enantiomers in fresh isolates of Plasmodium falciparum from East Africa.

Benflumetol, a novel antimalarial compound belonging to the fluorenes (2,3-benzindenes), has high blood schizontocidal activity, in vitro and in vivo, against mammalian plasmodia, including chloroquine-resistant Plasmodium falciparum. Due to its molecular structure benflumetol occurs in the dextrorotatory and the laevorotatory form. Normal synthesis yields the racemate of both enantiomers. Enantiomers of some antimalarial drugs possess different specific activity. It was therefore of interest to compare the response of P. falciparum to the enantiomers and the racemate of benflumetol in a variety of fresh, natural isolates. Measuring the concentration-specific inhibition of schizont maturation, the parallel investigation of 29 isolates produced no evidence of substantial activity differences between (+)-benflumetol, (-)-benflumetol and racemic benflumetol, the mean EC-50 values being 8.87, 9.71 and 12.44 nmol/l blood-medium-mixture, respectively.

High-harmonic generation and parametric amplification in the soft X-rays from extended electron trajectories.

We report, for the first time, the generation of high-order harmonics in a spectral range between 200 eV and 1 keV with an unusual spectral property: only every 4(th) (4i + 1, i∈ℵ) harmonic line appears, whereas the usual high-harmonic spectra consist of every odd (2i + 1) harmonic. We attribute this unique property to the quantum path interference of two extended electron trajectories that experience multiple re-scattering. In the well-established theory, electrons emitted via tunnel ionisation are accelerated by a laser field, return to the ion and recombine. The acceleration typically lasts for less than one optical cycle, and the electrons radiate in the extreme ultraviolet range at recombination. In contrast, for extended trajectories, electrons are accelerated over two or more optical cycles. Here, we demonstrate that two sets of trajectories dominate and provide substantial contributions to the generated soft X-ray radiation because they fulfil the resonance condition for X-ray parametric amplification.

Parametric amplification of attosecond pulse trains at 11†nm.

We report the first experimental demonstration of the parametric amplification of attosecond pulse trains at around 11 nm. The helium amplifier is driven by intense laser pulses and seeded by high-order harmonics pulses generated in a neon gas jet. Our measurements suggest that amplification takes place only if the seed pulse-trains are perfectly synchronized in time with the driving laser field in the amplifier. Varying the delay, we estimate the durations of the individual extreme ultraviolet pulses within the train to be on the order of 0.2 fs. Our results demonstrate that strong-field parametric amplification can be a suitable tool to amplify weak attosecond pulses from non-destructive pump-probe experiments and it is an important step towards designing amplifiers for realization of energetic XUV pulses with sub-femtosecond duration using compact lasers fitting in university laboratories.

New design of a multi-jet target for quasi phase matching.

An improved dual-gas quasi-phase matching (QPM) foil target for high harmonic generation (HHG) is presented. The target can be setup with 12 individual gas inlets each feeding multiple nozzles separated by a minimum distance of 10 µm. Three-dimensional gas density profiles of these jets were measured using a Mach-Zehnder Interferometer. These measurements reveal how the jets influence the density of gas in adjacent jets and how this leads to increased local gas densities. The analysis shows that the gas profiles of the jets are well defined up to a distance of about 300 µm from the orifice. This target design offers experimental flexibility, not only for HHG/QPM investigations, but also for a wide range of experiments due to the large number of possible jet configurations. We demonstrate the application to controlled phase tuning in the extreme ultraviolet using a 1 kHz-10 mJ-30 fs-laser system where interference between two jets in the spectral range from 17 to 30 nm was observed.

Velocity measurements in the middle cerebral arteries of healthy volunteers using 3D radial phase-contrast HYPRFlow: comparison with transcranial Doppler sonography and 2D phase-contrast MR imaging.

BACKGROUND AND PURPOSE: We have developed PC HYPRFlow, a comprehensive MRA technique that includes a whole-brain CE dynamic series followed by PC velocity-encoding, yielding a time series of high-resolution morphologic angiograms with associated velocity information. In this study, we present velocity data acquired by using the PC component of PC HYPRFlow (PC-VIPR). MATERIALS AND METHODS: Ten healthy volunteers (6 women, 4 men) were scanned by using PC HYPRFlow and 2D-PC imaging, immediately followed by velocity measurements by using TCD. Velocity measurements were made in the M1 segments of the MCAs from the PC-VIPR, 2D-PC, and TCD examinations. RESULTS: PC-VIPR showed approximately 30% lower mean velocity compared with TCD, consistent with other comparisons of TCD with PC-MRA. The correlation with TCD was r = 0.793, and the correlation of PC-VIPR with 2D-PC was r = 0.723. CONCLUSIONS: PC-VIPR is a technique capable of acquiring high-resolution MRA of diagnostic quality with velocity data comparable with TCD and 2D-PC. The combination of velocity information and fast high-resolution whole-brain morphologic angiograms makes PC HYPRFlow an attractive alternative to current MRA methods.

The effect of spatial resolution on wall shear stress measurements acquired using radial phase contrast magnetic resonance angiography in the middle cerebral arteries of healthy volunteers. Preliminary results.

We have recently implemented radial phase-contrast techniques that allow high resolution angiograms with velocity information to be acquired within clinically-useful imaging times. 10 healthy volunteers were scanned using PC-VIPR and PC-SOS, two high resolution phase-contrast techniques at spatial resolutions of 0.67×0.67×0.67 mm(3) and 0.4×0.4×1 mm(3) respectively. The velocity measurements from the two acquisitions were imported into a custom Matlab runtime environment that automatically calculated WSS values using Green's Theorem and B-spline interpolation. Time average axial WSS was 1.069 N/m(2) (95% confidence interval: 0.8628< x < 1.276) in the left and right middle cerebral arteries of the 10 healthy volunteers (n=20) when scanned by PC-VIPR, and 1.670 N/m(2) when scanned by PC-SOS (95% confidence interval: 1.395 < x < 1.946). This difference in means was statistically significant (p < 0.002). Previous investigators have found that higher spatial resolution results in higher WSS measurements because smaller voxel size results in fewer partial volume effects. This was true in our study as well. In this study, we found that PC-SOS has significantly higher spatial resolution than PC-VIPR and this followed in the WSS measurements. Higher in-plane spatial resolution allows WSS calculations to be performed more accurately because of increased precision near the vessel boundary.

Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4.

DAB389-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-related fusion protein which has been shown to be selectively toxic to cells expressing the mIL-4 receptor. In this report, we have used site-directed and in-frame deletion mutagenesis to study the role of the putative C-terminal alpha-helix (helix E) of the mIL-4 component of DAB389-mIL-4 in the intoxication process. We demonstrate that deletion of the C-terminal 15 amino acids of the fusion toxin leads to loss of cytotoxicity. The substitution of Phe496 with either Pro, Ala or Tyr, results in a greater than 20-fold decrease in cytotoxic activity of the respective mutant fusion toxins. In addition, substitution of Leu497 with either Ala or Glu results in a similar loss of cytotoxic activity. All of these mutant forms of the mIL-4 fusion toxin demonstrate a significant decrease in binding affinity (Ki) to the mIL-4 receptor in a competitive radioligand binding assay. In marked contrast, however, the substitution of Asp495 with Asn results in a 4-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro.

Early toxicity of recombinant interleukin-2 in cats.

Studies were conducted in cats to determine whether this species could serve as an animal model for the therapeutic toxicity of recombinant interleukin-2 in humans, and to establish the role of the area postrema in the causation of recombinant interleukin-2-induced vomiting. Injections of recombinant interleukin-2 (3.6 x 10(6) IU/kg, i.m.), given once every 24 hr for one to three days, evoked repeated vomiting in 4 out of 6 area postrema intact cats and in 3 out of 3 area postrema-ablated cats. These results suggest that the area postrema is not essential for the emetic action of recombinant interleukin-2. In anesthetized intact cats, no remarkable changes in ventilation, blood pressure, heart rate or blood pH were observed over 4.5 to 54 hr of continuous physiological recording after i.v. injections of recombinant interleukin-2 to a total dose as high as 27 x 10(6) IU/kg. Cat lymphocytes responded appropriately to the cytokinetic action of human recombinant interleukin-2.

Structural significance of the C-terminal amphiphilic helix of interleukin-2.

The structural significance of C-terminal amphiphilic alpha-helix of human interleukin-2 has been investigated using principles of protein design. Employing disulfide-mediated semi-synthesis, several multiple residue substitution patterns were studied in order to provide rapid insight into the most appropriate features to incorporate into fully recombinant proteins. Substitutions directed toward both stabilization and destabilization of the helix resulted in proteins with modulated bioactivity. Circular dichroism verified the conformational integrity and thermal stability of the derivatives. The biologic characteristics of each derivative were evaluated in the standard murine CTLL-2 assay and compared to activities exhibited in both human T-cell bioactivity and binding assay. A strategy for the design of protein ligand agonists and antagonists without knowledge of receptor contact residues is discussed.

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes.

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.

Monitoring of protein conformation by high-performance size-exclusion liquid chromatography and scanning diode array second-derivative UV absorption spectroscopy.

Genetic methods now allow the rapid production of mutant proteins for structure-function analysis. To properly interpret any change in biologic activity resulting from modification in primary sequence, it is essential to monitor conformational changes resulting from mutations. Several methods allow low-resolution protein conformational analysis. One method, second-derivative UV absorption spectroscopy, is particularly useful for proteins containing tyrosine and/or tryptophan residues. Using high-performance size-exclusion liquid chromatography and scanning diode array detection we have demonstrated that it is possible to monitor the degree of aggregation as well as conformational perturbation for a series of interleukin-2 structural mutants. Furthermore, the combination of high-performance liquid chromatography and second-derivative UV absorption spectroscopy avoids a potential artifactual contribution in non-chromatographic analysis due to protein aggregation.

Conformational perturbation of interleukin-2: a strategy for the design of cytokine analogs.

Interleukin-2 (IL-2) is a representative of a growing family of small proteins termed lymphokines which are responsible for mediating cell differentiation, growth and function in the immune system. Many of these proteins are being evaluated for their clinical potential. From the perspective of drug development, structure-function analysis of these molecules and their receptors require the use methodologies different than those traditionally employed for small peptides and other natural products. However, similar pharmacologic principles apply and an understanding of ligand-receptor interactions and the associated responses is required in order to efficiently pursue agonist and antagonist design. Although IL-2 is a protein of only 133 amino acid residues for which a low resolution X-ray structure does exist, the complexity of its receptor system has provided an added challenge to structure-function studies. Consequently, little is known concerning the receptor contact residues for this protein. We have attempted to utilize established principles of protein and peptide structure to manipulate the conformation of IL-2 in a manner which has provided analogs helpful for receptor interaction studies. These proteins have not only providing useful information on the nature of the IL-2 receptor but have also revealed potential strategies for the design of IL-2 agonists and antagonists.

A design approach to the structural analysis of interleukin-2.

A semi-synthetic protein design approach has been employed for the structural investigation of a putative helical region at the C-terminus of Interleukin-2. With crystallographic or NMR derived conformational data as yet unavailable, we have relied only on primary sequence information and computer-assisted modelling to direct the analysis. By employing both chemical peptide synthesis and recombinant DNA methods, the C-terminus of IL-2 was modified according to a strategy designed to stabilize helical secondary structure. A semi-synthetic protein incorporating 12 simultaneous amino acid replacements was constructed, which possessed potentiated biological activity and displayed a far UV circular dichroism spectrum comparable to a hybrid protein with the authentic sequence. By comparison, another hybrid protein containing a C-terminal region designed to contain helix breaking residues was totally devoid of bioactivity. These findings provide evidence that the modelling method correctly identified a helix necessary for the formation of a bioactive tertiary fold. Moreover, by employing semi-synthesis it was possible to circumvent the difficulties associated with the preparation, purification and analysis of multiple recombinant proteins, and also to avoid the unreliability of total chemical synthesis for proteins greater than 100 residues.