Ab initio excited-state dynamics of the photoactive yellow protein chromophore

The photoisomerization mechanism of the neutral form of the photoactive yellow protein (PYP) chromophore is investigated using ab initio quantum chemistry and first-principles nonadiabatic molecular dynamics (ab initio multiple spawning or AIMS). We identify the nature of the two lowest-lying excited states, characterize the short-time behavior of molecules excited directly to S2, and explain the origin of the experimentally observed wavelength-dependent photoisomerization quantum yield.     

Conformational analysis and electronic structure of acetanilide

The calculations of the electronic structure and conformational analysis of the acetanilide were carried out using the CNDO/2 method. The results show that the endo form is 1.2 Kcal/mole more stable than the exo form. The most stable conformation of the exo isomer corresponds to the dihedral angle of 90 ° between the phenyl and acetamide plane, whereas the minimum energy conformation of the endo isomer corresponds to the dihedral angle 50 °–60 °. A comparison of the calculated and experimental dipole moments suggests also the dihedral angle of 50 °–60 °. A comparison with experiment indicates that this molecular orbital method is good for conformational analysis and gives electronic structure which is compatible with spectroscopic measurement. The calculated conformational analysis and electronic structure of the acetanilide are in excellent agreement with experiments.We thank Professor J. P. Green for helpful discussions. This research was supported by a grant from the National Institute of Mental Health (MH-17489-01).     

Phage-display selection of a human single-chain fv antibody highly specific for melanoma and breast cancer cells using a chemoenzymatically synthesized G(M3)-carbohydrate antigen

Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 10(-5) M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.     

Peak compression effects in capillary electrochromatography of basic drug substances using a strong cation-exchanger

Peak compression effects in capillary electrochromatography of basic drug substances using a strong cation-exchanger have been studied. Extremely narrow peaks with apparent efficiencies of several million plates per meter could be obtained when the composition of the sample zone differed from that of the mobile phase. The increased efficiencies were predominately observed when the analyte had an elution time similar to that of the electroosmotic flow marker. Peak compression was found to be reproducible and could be obtained for all investigated basic drug substances by altering the composition of the mobile phase in such a way that the analyte co-eluted with the sample zone. An explanation of the observed phenomena is proposed. A sample zone differing in composition from the mobile phase will disturb the equilibrium between the stationary and mobile phase. The elution rate of an analyte will consequently be different when residing inside the sample zone. If the analyte migrates through the sample zone at a higher speed than the rest of the mobile phase and is strongly retained after passing through a boundary in the sample zone, a continuous stacking can be obtained trapping the analyte as a very narrow band.     

Preparation of La0.75Sr0.25MnO3 Nano-Phase Powders and Films from Alkoxide Precursors

The first purely alkoxide-based sol-gel route to nano-phase powders and thin films of perovskite La_0.75Sr_0.25MnO₃ is described. The phase and microstructure evolution on heat treatment of free gel films to form the target nano-phase oxide were investigated by TGA, IR spectroscopy, powder XRD, SEM and TEM-EDS. The xerogel consisted of a hydrated oxo-carbonate, without remaining alkoxo groups or solvent. Heating at 5°C·min^–1 decomposed the carbonate groups and yielded the pure perovskite La_0.75Sr_0.25MnO₃ at 760°C. The cell dimensions were virtually unchanged from the first observation of perovskite at 680°C, to 1000°C, 4 h. The monoclinic cell of La_0.75Sr_0.25MnO₃ obtained at 1000°C, 4 h, had the dimensions a = 5.475(1), b = 5.504(2), c = 7.771(1) Å, = 90.50(2), fitting the literature data quite well. Crack-free, homogenous, 150 nm thick La_0.75Sr_0.25MnO₃ films were prepared by spin-coating Si/SiO₂/TiO₂/Pt and polycrystalline -Al₂O₃ substrates with a 0.6 M alkoxide solution, followed by heating at 5°C·min^–1 to 800°C, 30 min.     

Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities

Hydrophilic interaction chromatography (HILIC) is described as a useful alternative to reversed-phase chromatography for applications involving polar compounds. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal-phase retention behavior. Silica and amino columns with aqueous-acetonitrile mobile phases offer potential for use in the HILIC mode. An examination of the retention and separation of several pyrimidines, purines, and amides on silica and amino columns from three manufacturers revealed that mobile phases should contain a buffer or acid for pH control to achieve similar and reproducible results among columns from different sources. Amino columns may also be used in an anion-exchange mode, which provides an advantage for some applications. In some cases, silica can provide different selectivity and better separation than an amino column. Example applications include: low-molecular-mass organic acids and amides as impurities in non-polar drug substances, 5-fluorouracil in 5-fluorocytosine, guanine in acyclovir, and different selectivity for polar basic compounds compared to an ion-pairing system.     

Effectiveness of antioxidants: Suppression of evolution of gaseous degradation products from low-density polyethylene during thermo-oxidation

The present investigation is part of a project concerning improvements of the working environment for polymer manufacturing processes at high temperatures; for example, welding and extrusion coating.The evolution of gaseous degradation products from unstabilized and antioxidant stabilized low-density polyethylene during thermo-oxidation in air at 210°C has been studied by a simple technique using a modified gas chromatograph. Of the thirteen antioxidants investigated (phenols, phosphites, phosphonites, sulphides and metal dithiocarbamates), only phenols (radical scavengers) effectively suppress the formation of gaseous degradation products from the polymer during an induction period.The gaseous degradation products were analysed with combined gas chromatography-mass spectrometry (GC-MS), using direct trapping on the GC column (Porapak® QS). Apart from water and carbon dioxide, the major products are formaldehyde, acetaldehyde, formic and acetic acids. The addition of effective antioxidants strongly reduces the absolute amounts of the degradation products during the induction period, but does not change their relative quantities.     

Determination of airborne isocyanates as di-n-butylamine derivatives using liquid chromatography and tandem mass spectrometry

A method for the determination of isocyanates as di-n-butyl amine (DBA) derivatives using tandem mass spectrometry (MS/MS) and electrospray ionisation (ESI) is presented. Multiple-reaction monitoring (MRM) of the protonated molecular ions and corresponding deuterium-labelled d₉-DBA derivatives resulted in selective quantifications with correlation coefficients >0.998 for the DBA derivatives of isocyanic acid (ICA), methyl isocyanate (MIC), ethyl isocyanate (EIC), propyl isocyanate (PIC), phenyl isocyanate (PhI), 1,6-hexamethylene diisocyanate (HDI), 2,4-, 2,6-toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), 4,4′-methylenediphenyl diisocyanate (MDI), 3-ring MDI, 4-ring MDI, HDI-isocyanurate, HDI-diisocyanurate, HDI-biuret and HDI-dibiuret. The instrumental precision for 10 repeated injections of a solution containing 0.1 μg ml⁻¹ of the studied derivatives was <2%. Performing MRM of the product ion [DBA + H]⁺ (m/z = 130) from the protonated molecular ion resulted in the lowest detection limits, down to 10 amol (for TDI). Quantification of concentrations below 10⁻⁶ of the occupational exposure limit (OEL) for TDI during 10 min of air sampling was made possible. In an effort to control the formation of alkali adducts, addition of lithium acetate to the mobile phase and monitoring of lithium adducts was evaluated. Having lithium present in the mobile phase resulted in complete domination of [M + Li]⁺ adducts, but detection limits for the studied compounds were not improved. Different deuterium-labelled derivatives as internal standards were evaluated. (1) DBA derivatives of deuterium-labelled isocyanates (d₄-HDI, d₃-2,4-TDI, d₃-2,6-TDI and d₂-MDI), (2) d₉-DBA derivatives of the corresponding isocyanates and (3) d₁₈-DBA derivatives of the corresponding isocyanates. An increase in number of deuterium in the molecule of the internal standard resulted in an increase in instrumental precision and a decrease in correlation within calibration series.     

EARLY CELL KINETIC EFECTS OF A SINGLE DOSE OF NARROW-BANDED ULTRAVIOLET B IRRADIATION ON THE RAT CORNEAL EPITHELUM

Abstract— The right eyes of 40 rats were exposed to a signal erythemogenic dose fo ultraviolet B irradiation (UVB) at 297nm. The irradiation was directed perpenddicualr to the center of the cornea. The left eyes served as controls. The animals were randomly assigned into 10 groups. The labelling index (LI) after pluse labeling the tritiated thymidine and the mitotic rate (MR) after Colcemid administration were registered in the corneal epithelium at predetermined intervals up to 96 h after the irradiation. A mathematical method was used to corealted corresponding corneal areas from the different animals. In the central the LI was considerably reduced up to 36h after the irradiation. The LI increased toward the peripheral cornea and reached normal values at the limbal area. The MR was also reduced up to 36h. However, this reduction was over the entire epithelium. The block in cell proliferation was followed by increased proliferation.