Mass spectrometry guided in situ proteolysis to obtain crystals for X-ray structure determination

A strategy for increasing the efficiency of protein crystallization/structure determination with mass spectrometry has been developed. This approach combines insights from limited proteolysis/mass spectrometry and crystallization via in situ proteolysis. The procedure seeks to identify protease-resistant polypeptide chain segments from purified proteins on the time-scale of crystal formation, and subsequently crystallizing the target protein in the presence of the optimal protease at the right relative concentration. We report our experience with 10 proteins of unknown structure, two of which yielded high-resolution X-ray structures. The advantage of this approach comes from its ability to select only those structure determination candidates that are likely to benefit from application of in situ proteolysis, using conditions most likely to result in formation of a stable proteolytic digestion product suitable for crystallization.     

Linear expanded xanthines

Expansion of the xanthine ring system has been accomplished by linear formation of a benzo, pyrido or pyrazino ring between the pyrimidine and imidazole portions.

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Linear expandierte Xanthine (Kurze Mitteilung)

Zusammenfassung Durch Einbau eines Benzo-, Pyrido- oder Pyrazino-Ringes zwischen den Pyrimidin- und Imidazoleinheiten wurde die lineare Expansion des Xanthin-Ringsystems erreicht.

     

Repeated-load testing of schist and limestone aggregate materials used in unbound forest roads

This study examined the behaviour of two completion aggregates that were placed on a silty sandy formation material and subjected to repeated loading in a large-scale testing rig. The completion aggregates were (i) a good quality limestone, and (ii) a poor quality schist, and are used in the construction of unbound forest roads in Ireland. The schist was tested on its own and in combination with the completion limestone aggregate for up to 150,000 load applications with a maximum loading pressure of 1000 kPa. Measurements of surface deflections and formation pressures were made during each test. A combination of a 200 mm layer of the limestone aggregate placed on top of a 250 mm layer of the schist aggregate produced the lowest permanent deformations, resilient deflections, and resilient pressures under dry and wet undrained conditions.     

Determination of chlorantraniliprole residues in crops by liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry/mass spectrometry

An analytical method is presented for the determination of chlorantraniliprole residues in crops. Chlorantraniliprole residues were extracted from crop matrixes with acetonitrile after a water soak. The extracts were passed through a strong anion-exchange (SAX) SPE cartridge stacked on top of a reversed-phase (RP) polymer cartridge. After both cartridges were rinsed and vacuum-dried, the SAX cartridge was removed, and chlorantraniliprole was eluted from the RP polymer cartridge with acetonitrile. The acetonitrile eluate was evaporated to dryness, reconstituted, and analyzed using an LC/MS/MS instrument equipped with an atmospheric pressure chemical ionization source. The method was successfully validated at 0.010, 0.10, and 10 mg/kg for the following crop matrixes: potatoes, sugar beets (tops), lettuce, broccoli, soybeans, soybean forage, tomatoes, cucumbers, oranges, apples, pears, peaches, almonds (nutmeat), rice grain, wheat grain, wheat hay, corn stover, alfalfa forage, cottonseed, grapes, and corn grain. The average recoveries from all crop samples fortified at the method LOQ ranged from 91 to 108%, with an overall average recovery of 97%. The average recoveries from all crop samples fortified at 10 times the method LOQ ranged from 89 to 115%, with an overall average recovery of 101%. For all of the fortified control samples analyzed in this study, the overall average recovery was 99%.     

Double k-sets in symplectic generalized quadrangles

In classical projective geometry, a double six of lines consists of 12 lines ? ₁, ? ₂, . . . , ? ₆, m ₁, m ₂, . . . , m ₆ such that the ? _i are pairwise skew, the m _i are pairwise skew, and ? _i meets m _j if and only if i ≠ j. In the 1960s Hirschfeld studied this configuration in finite projective spaces PG(3, q) showing they exist for almost all values of q, with a couple of exceptions when q is too small. We will be considering double-k sets in the symplectic geometry W(q), which is constructed from PG(3, q) using an alternating bilinear form. This geometry is an example of a generalized quadrangle, which means it has the nice property that if we take any line ? and any point P not on ?, then there is exactly one line through P meeting ?. We will discuss all of this in detail, including all of the basic definitions needed to understand the problem, and give a result classifying which values of k and q allow us to construct a double k-set of lines in W(q).     

Irradiation of Amelanotic Melanoma Cells with 532 nm High Peak Power Pulsed Laser Radiation in the Presence of the Photothermal Sensitizer Cu(II)-Hematoporphyrin: A New Approach to Cell Photoinactivation

Cu(II)-hematoporphyrin (CuHp) was efficiently accumulated by B78H1 amelanotic melanoma cells upon incubation with porphyrin concentrations up to 52 microM. When the cells incubated for 18 h with 13 microM CuHp were irradiated with 532 nm light from a Q-switched Nd: YAG laser operated in a pulsed mode (10 ns pulses, 10 Hz) a significant decrease in cell survival was observed. The cell photoinactivation was not the consequence of a photodynamic process, as CuHp gave no detectable triplet signal upon laser flash photolysis excitation and no decrease in cell survival was observed upon continuous wave irradiation. Thus, it is likely that CuHp sensitization takes place by photothermal pathways. The efficiency of the photoprocess was modulated by different parameters; thus, while varying the amount of added CuHp in the 3.25-26 microM range had little effect, pulse energies larger than 50 mJ and irradiation times of at least 120 s were necessary to induce a cell inactivation of about 50%. The porphyrin-cell incubation time prior to irradiation had a major influence on cell survival, suggesting that the nature of the CuHp microenvironment can control the efficiency of photothermal sensitization.     

GLASS and PLASTIC PHOTOLUMINESCENCE: INTERACTION WITH AQUEOUS MEDIA and CULTURED MAMMALIAN CELLS

Several types of glass and plastic materials were shown to exhibit intense photoluminescence when irradiated with UV. Water or phosphate buffered saline (PBS) contained within vessels of the respective materials when irradiated, also demonstrated relatively long-lived luminescence. A significant percentage (30%) of cultured mammalian cells were killed when exposed to UV-irradiated glass beads. The nature of the luminescence of water or PBS, or whether this or the photoluminescence of glass is directly responsible for cell toxicity, is unknown. However, we call attention to this phenomenon as a potential complicating factor in photobiological studies.     

Femtosecond fabrication of waveguide-like micro-structures in a photorefractive polymer

In this letter, we report on, for the first time, the successful femtosecond micro-fabrication of continuous waveguide-like channels in the photorefractive polymer consisting of the nonlinear chromophore 2,5-dimethyl-4-(p-nitrophenylazo)anisole (DMNPAA), the photosensitive compound 2,4,7-trinitro-9-flourenone (TNF), and the plasticiser N-ethylcarbazole (ECZ) all doped in the polymer matrix poly (methyl methacry-late) (PMMA). These channels are caused by the change in refractive index as a result of the localised heating of the polymer and therefore have an important potential for micro-photonic devices in future.     

Influence of Linkage Stereochemistry and Protecting Groups on Glycosidic Bond Stability of Sodium Cationized Glycosyl Phosphates

Energy-resolved collision-induced dissociation (ER-CID) experiments of sodium cationized glycosyl phosphate complexes, [GP _x +Na]⁺, are performed to elucidate the effects of linkage stereochemistry (α versus β), the geometry of the leaving groups (1,2-cis versus 1,2-trans), and protecting groups (cyclic versus non-cyclic) on the stability of the glycosyl phosphate linkage via survival yield analyses. A four parameter logistic dynamic fitting model is used to determine CID_50% values, which correspond to the level of rf excitation required to produce 50% dissociation of the precursor ion complexes. Present results suggest that dissociation of 1,2-trans [GP _x +Na]⁺ occurs via a McLafferty-type rearrangement that is facilitated by a syn orientation of the leaving groups, whereas dissociation of 1,2-cis [GP_x+Na]⁺ is more energetic as it involves the formation of an oxocarbenium ion intermediate. Thus, the C1?C2 configuration plays a major role in determining the stability/reactivity of glycosyl phosphate stereoisomers. For 1,2-cis anomers, the cyclic protecting groups at the C4 and C6 positions stabilize the glycosidic bond, whereas for 1,2-trans anomers, the cyclic protecting groups at the C4 and C6 positions tend to activate the glycosidic bond. The C3 O-benzyl (3 BnO) substituent is key to determining whether the sugar or phosphate moiety retains the sodium cation upon CID. For 1,2-cis anomers, the 3 BnO substituent weakens the glycosidic bond, whereas for 1,2-trans anomers, the 3 BnO substituent stabilizes the glycosidic bond. The C2 O-benzyl substituent does not significantly impact the glycosidic bond stability regardless of its orientation.

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Graphical abstract ?

     

Infrared Multiple Photon Dissociation Action Spectroscopy and Theoretical Studies of Diethyl Phosphate Complexes: Effects of Protonation and Sodium Cationization on Structure

The gas-phase structures of deprotonated, protonated, and sodium-cationized complexes of diethyl phosphate (DEP) including [DEP − H]⁻, [DEP + H]⁺, [DEP + Na]⁺, and [DEP − H + 2Na]⁺ are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy using tunable IR radiation generated by a free electron laser, a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) with an electrospray ionization (ESI) source, and theoretical electronic structure calculations. Measured IRMPD spectra are compared to linear IR spectra calculated at the B3LYP/6-31G(d,p) level of theory to identify the structures accessed in the experimental studies. For comparison, theoretical studies of neutral complexes are also performed. These experiments and calculations suggest that specific geometric changes occur upon the binding of protons and/or sodium cations, including changes correlating to nucleic acid backbone geometry, specifically P–O bond lengths and ∠OPO bond angles. Information from these observations may be used to gain insight into the structures of more complex systems, such as nucleotides and solvated nucleic acids.