Ray-tracing simulation of the radiation dose distribution on the surface of the spherical phantom of the MATROSHKA-R experiment onboard the ISS.

Space radiation is one of the main concerns for human space flights. The prediction of the radiation dose for the actual spacecraft geometry is very important for the planning of long-duration missions. We present a numerical method for the fast calculation of the radiation dose rate during a space flight. We demonstrate its application for dose calculations during the first and the second sessions of the MATROSHKA-R space experiment with a spherical tissue-equivalent phantom. The main advantage of the method is the short simulation time, so it can be applied for urgent radiation dose calculations for low-Earth orbit space missions. The method uses depth-dose curve and shield-and-composition distribution functions to calculate a radiation dose at the point of interest. The spacecraft geometry is processed into a shield-and-composition distribution function using a ray-tracing method. Depth-dose curves are calculated using the GEANT4 Monte-Carlo code (version 10.00.P02) for a double-layer aluminum-water shielding. Aluminum-water shielding is a good approximation of the real geometry, as water is a good equivalent for biological tissues, and aluminum is the major material of spacecraft bodies. The method is applied to model the dose distribution on the surface of the spherical phantom in the MATROSHKA-R space experiment. The experiment has been carried out onboard the ISS from 2004 to the present. The absorbed dose was determined in 32 points on the phantom's surface. We find a good agreement between the data obtained in the experiment and our calculation results. The simulation method is thus applicable for future radiation dose predictions for low-Earth orbit missions and experiments.

Changing the ortho/para ratio in aromatic acylation reactions by changing reaction conditions: a mechanistic explanation from kinetic measurements.

Kinetic measurements of the acylation of toluene (2a) and p-xylene (2b), side-chain deuterated toluene (2a-d(3)), as well as perdeuterated toluene (2a-d(8)) and p-xylene (2b-d(10)) with the aroyl triflate 1 in 1,2-dichloroethane reveal a strong dependence of the isotope effect on reaction conditions. In the presence of trifluoromethanesulfonic acid (HOTf), the second-order rate constants k(H)/k(D) observed are in the order of 1.75-1.94, whereas in the presence of 2,4,6-tri-tert-butylpyridine (4) rate constants k(H)/k(D) of 1.14-1.25 are found. The primary kinetic isotope effects observed correlate with the ortho/para ratio of the acylation of toluene. In the presence of 4 a relatively high percentage ( approximately 30%) of ortho product is obtained, whereas under acidic conditions the ratio is only 10%. The correlation between isotope effects and isomer distributions is obviously due to the rate of deprotonation of the corresponding sigma-complex intermediates. Assuming a bent structure for sigma-complexes, the conformation giving deprotonation is preferred in the para sigma-complex in comparison with ortho complex.

Mechanistic aspects of cyanogenesis from active-site mutant Ser80Ala of hydroxynitrile lyase from Manihot esculenta in complex with acetone cyanohydrin.

The structure and function of hydroxynitrile lyase from Manihot esculenta (MeHNL) have been analyzed by X-ray crystallography and site-directed mutagenesis. The crystal structure of the MeHNL-S80A mutant enzyme has been refined to an R-factor of 18.0% against diffraction data to 2.1-A resolution. The three-dimensional structure of the MeHNL-S80A-acetone cyanohydrin complex was determined at 2.2-A resolution and refined to an R-factor of 18.7%. Thr11 and Cys81 involved in substrate binding have been substituted by Ala in site-directed mutagenesis. The kinetic measurements of these mutant enzymes are presented. Combined with structural data, the results support a mechanism for cyanogenesis in which His236 as a general base abstracts a proton from Ser80, thereby allowing proton transfer from the hydroxyl group of acetone cyanohydrin to Ser80. The His236 imidazolium cation then facilitates the leaving of the nitrile group by proton donating.

Structure of hydroxynitrile lyase from Manihot esculenta in complex with substrates acetone and chloroacetone: implications for the mechanism of cyanogenesis.

The crystal structures of hydroxynitrile lyase from Manihot esculenta (MeHNL) complexed with the native substrate acetone and substrate analogue chloroacetone have been determined and refined at 2.2 A resolution. The substrates are positioned in the active site by hydrogen-bond interactions of the carbonyl O atom with Thr11 OG, Ser80 OG and, to a lesser extent, Cys81 SG. These studies support a mechanism for cyanogenesis as well as for the stereospecific MeHNL-catalyzed formation of (S)-cyanohydrins, which closely resembles the base-catalyzed chemical reaction of HCN with carbonyl compounds.

Hydroxynitrile lyases in stereoselective catalysis.

(R)- as well as (S)-cyanohydrins are now easily available as a result of the excellent accessibility, the relatively high stability and the easy handling of hydroxynitrile lyases (HNLs). The optimization of reaction conditions (solvent, temperature, and using site-directed mutagenesis, etc.) has enabled HNL-catalyzed preparations of optically active cyanohydrins on a technical scale. The enantioselectivity of chiral metal-complex-catalyzed additions of trimethylsilyl cyanide to aldehydes has been improved, but is, by far, not yet competitive with the HNL-catalyzed reactions.

A convenient synthesis of optically active 5,5-disubstituted 4-amino- and 4-hydroxy-2(5H)-furanones from (S)-ketone cyanohydrins.

(S)-Ketone cyanohydrins (S)-2 are accessible by enantioselective HCN addition to ketones 1 by using hydroxynitrile lyase from Manihot esculenta ((S)-MeHNL) as a biocatalyst. Acylation of (S)-2 gave the corresponding (S)-acyloxynitriles (S)-3, which can be cyclized by LHMDS to give 5,5-disubstituted (S)-4-amino-2(5H)-furanones (S)-4 and (S)-5. Different substituents (H. Me, OBn, OH) in the 3-position of the furanones were introduced by selecting the appropriate acylating agent, which in the case of benzyloxyacetyl chloride led to the novel structure type of 4-amino-3-hydroxyfuranones (S)-5. For the synthesis of 5,5-disubstituted (S)-tetronic acids (S)-8, ketone cyanohydrins (S)-2 were first transformed into the corresponding 2-hydroxy esters (S)-6. Acylation of (S)-6 gave 2-acyloxy esters (S)-7, which, by treatment with LHMDS or LDA, afforded tetronic acids (S)-8 in high yields and enantiomeric excesses. By debenzylation of benzyloxy acetoxy derivatives (S)-8e,f, the new vitamin C analogues (S)-9a,b were generated. All the described tetronic acid and aminofuranone derivatives were obtained in good chemical yields and without racemization with respect to the starting cyanohydrins (S)-2. In many cases the enantiomeric purity could be enriched by simple recrystallization (e.g. (S)-4a from 69% ee to > 99% ee).

Mimicry of human IgE epitopes by anti-idiotypic antibodies.

According to Jerne's network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunization, and confirm Jerne's hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.

Crystallization and preliminary x-ray diffraction analysis of hydroxynitrile lyase from cassava (Manihot esculenta).

Hydroxynitrile lyase from M. esculenta (cassava) was crystallized in two different crystal forms by the hanging-drop vapour-diffusion method. Crystals of form I were obtained from a mixture of polyethylene glycol 8000 and 2-methyl-2,4-pentanediol, and belong to the tetragonal space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 105.9, c = 188.9 A and with two molecules in the asymmetric unit. These crystals diffract to 2.9 A with conventional X-ray sources and beyond 2.1 A resolution with synchrotron radiation. The crystals are relatively sensitive to radiation damage and conditions for flash-cooling the crystals have been established. A complete native data set has been collected up to 2.2 A resolution. Crystal form II has been obtained at pH 5.6 using lithium sulfate as a precipitant. The crystals apparently belong to the orthorhombic space group P21212, with unit-cell parameters a = 117.52, b = 127.09 and c = 78.08 A, have two molecules in the asymmetric unit and diffract to beyond 2.0 A resolution. A complete native data set has been collected to 2.2 A resolution.

Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor.

A panel of monoclonal antibodies (8H10/D11, 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, 8A4/G12/F9, and 8H10/F12) was raised in mice against the recombinant 20-kDa extracellular part of the alpha-chain of the human IgE high affinity receptors (ecFc epsilon RIalpha) produced in insect cells. The antibodies secreted by hybridomas were selected for specific binding to ecFc epsilon RIalpha, by enzyme-linked immunosorbent assay (ELISA). The selected clones were further characterized in surface plasmon resonance (SPR) experiments with ecFc epsilon RIalpha covalently immobilized on the surface of a sensor chip. The generated hybridomas can be divided into three groups. Hybridoma supernatants 8A4/G12/F9 and 8H10/F12 inhibited binding of human IgE to immobilized ecFc epsilon RIalpha in SPR (Group 1). Isotyping revealed that 8A4/G12/F9 and 8H10/F12 were of the IgE/kappa type. Antibodies present in the remaining supernatants were noninhibitory and bound to ecFc epsilon RIalpha in ELISA with intensities comparable to each other. Isotype analysis of antibodies secreted by these hybridomas showed that the antibodies 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, and 8H10/D11 were IgG1/kappa. The hybridoma supernatants were purified via protein A chromatography. In a SPR experiment, ecFc epsilon RIalpha, displayed by immobilized human IgE, was still recognized by 6F9/H8 and 6F9/G9 (Group 2) as expected for noninhibitory antibodies. Surprisingly, 8H10/D11, 5F2/F8/H11, and 5F2/F8/G10 (Group 3) did not bind to this complex although they do not inhibit the binding of human IgE to ecFc epsilon RIalpha. All purified monoclonal antibodies gave positive signals in Western blotting.

Hydroxynitrile lyases of higher plants.

Release of HCN from cyanogenic glycosides is due to the cleavage of the carbohydrate moiety by beta-glucosidases to yield the corresponding alpha-hydroxynitrile, which dissociates spontaneously into HCN and a carbonyl compound, or by action of an alpha-hydroxynitrile lyase (HNL). A short review of the regulation of the catabolism of cyanogenic glycosides during cyanogenesis and germination of cyanogenic plants is given. The major biochemical properties of HNLs purified from various species of higher plants are summarized. Thereafter the phylogenetic relationship, molecular structure and catalytic mechanism of these enzymes are discussed. Finally we give an overview of recent progress in the use of HNLs as biocatalysts for the synthesis of optically active alpha-hydroxynitriles which are important building blocks in the fine chemical and pharmaceutical industries.

Crystallographic studies and preliminary X-ray investigation of (S)-p-hydroxy-mandelonitrile lyase from Sorghum bicolor (L.).

(S)-p-Hydroxy-mandelonitrile lyase from Sorghum bicolor has been crystallized in three different forms using the hanging-drop vapor-diffusion technique. Crystal form I is obtained from 1.4 M (NH(4))(2)SO(4) in 100 mM Na-acetate, pH 4.6, and belongs to the orthorhombic space group P2(1)2(1)2(1). The cell dimensions are a = 71.4, b = 95.8, c = 149.1 A. A complete set of diffraction data has been collected to 2.6 A resolution. Form II crystals are grown from 500 mM Li(2)SO(4) in 13% polyethylene glycol 8000. These crystals appear as hexagonal plates and diffract to 2.98 A resolution but apparently are twinned. Cocrystallizing hydroxynitrile lyase with the inhibitor benzoic acid using 1.4 M (NH(4))(2)SO(4) in 100 mM Na citrate, pH 5.4 as precipitant yields crystal form III, which belongs to the monoclinic space group C2 with a = 150.7, b = 103.7, c = 90.6 A, beta = 101.3. X-ray diffraction data were collected to 2.3 A resolution.

Function and regulation of Fc epsilon RI expression on monocytes from non-atopic donors.

BACKGROUND: The high affinity receptor for IgE (Fc epsilon RI) has recently been identified on antigen presenting cells, i.e. Langerhans cells and monocytes from atopic donors and it was hypothesized that Fc epsilon RI expression levels correlated with allergy. OBJECTIVE: The aims of the study was to investigate the function and expression of Fc epsilon RI on monocytes from non-atopic donors. METHODS: Purified monocytes or peripheral blood mononuclear cells were used to study Fc epsilon RI expression and signal transduction on CD14 positive cells by flow cytometry and/or confocal laser microscopy. RESULTS: Freshly isolated monocytes from healthy individuals (n = 58) were shown to express Fc epsilon RI (median 18%, range 2-66%). No IgE was bound to these receptors in vivo, and in vitro no significant binding of complete IgE molecules could be obtained. IgE positive monocytes from atopic donors were also found to have free Fc epsilon RI incapable of binding IgE in vitro. On all CD14 positive cells free Fc epsilon RI expression was rapidly and completely lost during culture in conventional culture media (IMDM, RPMI) but not in phosphate buffered saline (PBS). Moreover, signal transduction through free Fc epsilon RI appeared to be inhibited. However, both IgE binding and calcium mobilization were restored by treatment of fresh non-atopic monocytes with neuraminidase. Importantly, culturing these monocytes overnight in conventional medium containing 2 micrograms/mL IgE induced a cycloheximide insensitive accumulation of IgE bound to Fc epsilon RI and, in addition, led to cell activation. CONCLUSION: Monocytes from both atopic donors and healthy individuals express Fc epsilon RI, but the previously reported different expression levels between the two groups seem to be directly related to the absence or presence of IgE in the serum. This may be due to the fact that Fc epsilon RI is subjected to a constant turnover process which is slowed down but not prevented by ligand binding. In addition, free Fc epsilon RI on non-atopic monocytes are under control of a neuramindase sensitive structure(s), which influences signal transduction and IgE binding.

Modal analysis of noise in signal-processing-in-the-element detectors.

Detector noise limits the performance of signal-processing-in-the-element detectors. For detectors to be optimized, an expression for the signal and noise must be found. The results of the eigenmode solution to the charge transport problem are used to derive the power spectral density of the noise in analytic form. This result is then coordinated with a similarly obtained modulation transfer function to yield a frequency-dependent signal-to-noise ratio (SNR). The SNR is used to reveal performance trends over several ranges of detector parameters. The most important result is that the contact boundary velocity strongly controls the SNR. The optimum SNR condition occurs when the contacts are not perfectly ohmic but exhibit a partially blocking behavior.

Modulation-transfer-function-enhanced readout for SPRITE detectors.

A new readout structure is investigated for signal-processing-in-the-element detectors that yields a modulation transfer function that is 3.5 dB better than those currently used. Experimental verification is performed in Si rather than HgCdTe, with similarity relations derived for the two semiconductors.

Purification and Characterization of a Novel (R)-Mandelonitrile Lyase from the Fern Phlebodium aureum.

Using high-performance liquid chromatography and nuclear magnetic resonance we identified vicianin as the cyanogenic compound of Phlebodium aureum. The (R)-hydroxynitrile lyase involved during cyanogenesis in the catabolism of the aglycon ([R]-mandelonitrile) was purified to apparent homogeneity. The purified holoenzyme is a homomultimer with subunits of Mr = 20,000. At least three isoforms of the enzyme exist. In contrast to other hydroxynitrile lyases, mandelonitrile lyase (MDL) from P. aureum was not inhibited by sulfhydryl- or hydroxyl-modifying reagents, suggesting a different catalytic mechanism. The enzyme is active over a broad temperature range, with maximum activity between 35 and 50[deg]C, and a pH optimum at 6.5. In contrast to (R)-MDLs isolated from several species of the Rosaceae family, (R)-MDL from P. aureum is not a flavoprotein. The substrate specificity was investigated using immobilized enzyme and diisopropyl ether as solvent. The addition of cyanide to aromatic and heterocyclic carbonyls is catalyzed by this (R)-MDL, whereas aliphatic carbonyls are poorly converted.

Modal analysis of transport processes in SPRITE detectors.

Carrier transport in signal-processing-in-the-element (SPRITE) detectors is an important phenomenon because it determines properties such as the responsivity and the modulation transfer function (MTF). The previous literature has presented approximate solutions to the transport problem that neglect boundary effects, which have long been thought to play a major role in SPRITE behavior. We present a new solution to the problem through the use of modal analysis. This method intrinsically includes boundary conditions and thus is more complete than the previous analysis. Furthermore we use this solution to derive expressions for the MTF. The effects of the boundary conditions on the MTF are studied to determine their optimum values.

Crystallization and preliminary X-ray diffraction studies of mandelonitrile lyase from almonds.

Single crystals of three different isoenzymes of (R)-(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2(1) with unit cell parameters a = 69.9, b = 95.1, c = 95.6 A, and beta = 118.5 degrees. A complete set of diffraction data has been collected to 2.6 A resolution on native crystals of isoenzyme III.