Radiation damage reveals promising interaction position

High‐resolution structural data of protein inhibitor complexes are the key to rational drug design. Synchrotron radiation allows for atomic resolutions but is frequently accompanied by radiation damage to protein complexes. In this study a human aldose reductase mutant complexed with a bromine‐substituted inhibitor was determined to atomic resolution [Protein Data Bank (PDB) code 3onc ]. Though the radiation dose was moderate, a selective disruption of a bromine–inhibitor bond during the experiment was observed while the protein appears unaffected. A covalent bond to bromine is cleaved and the displaced atom is not scattered throughout the crystal but can most likely be assigned as a bromide to an additional difference electron density peak observed in the structure. The bromide relocates to an adjacent unoccupied site where promising interactions to protein residues stabilize its position. These findings were verified by a second similar structure determined with considerably higher radiation dose (PDB code 3onb ).     

Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.     

Photolysis of (3-methyl-2H-azirin-2-yl)-phenylmethanone: direct detection of a triplet vinylnitrene intermediate

The photoreactivity of (3-methyl-2H-azirin-2-yl)-phenylmethanone, 1, is wavelength-dependent (Singh et al. J. Am. Chem. Soc. 1972, 94, 1199-1206). Irradiation at short wavelengths yields 2P, whereas longer wavelengths produce 3P. Laser flash photolysis of 1 in acetonitrile using a 355 nm laser forms its triplet ketone (T(1K), broad absorption with λ(max) ~ 390-410 nm, τ ~ 90 ns), which cleaves and yields triplet vinylnitrene 3 (broad absorption with λ(max) ~ 380-400 nm, τ = 2 μs). Calculations (B3LYP/6-31+G(d)) reveal that T(1K) of 1 is located 67 kcal/mol above its ground state (S(0)) and has a long C-N bond (1.58 ?), and the calculated transition state to form 3 is only 1 kcal/mol higher in energy than T(1K) of 1. The calculations show that 3 has significant 1,3-carbon iminyl biradical character, which explains why 3 reacts efficiently with oxygen and decays by intersystem crossing to the singlet surface. Photolysis of 1 in argon matrixes at 14 K produced ketene imine 7, which presumably is formed from 3 intersystem crossing to 7. In comparison, photolysis of 1 in methanol with a 266 nm laser produces mainly ylide 2 (λ(max) ~ 380 nm, τ ~ 6 μs, acetonitrile), which decays to form 2P. Ylide 2 is formed via singlet reactivity of 1, and calculations show that the first singlet excited state of the azirine chromophore (S(1A)) is located 113 kcal/mol above its S(0) and that the singlet excited state of the ketone (S(1K)) is 85 kcal/mol. Furthermore, the transition state for cleaving the C-C bond in 1 to form 2 is located 49 kcal/mol above the S(0) of 1. Thus, we theorize that internal conversion of S(1A) to a vibrationally hot S(0) of 1 forms 2, whereas intersystem crossing from S(1K) to T(1K) results in 3.     

Thermogravimetric analysis of photodegraded acrylic coated lime wood

A series of experiments were carried out to investigate the photodegradation of lime wood (Tilia cordata Mill.) coated with acrylic copolymer during artificial UV/Vis light irradiation for 600 h. Photodegradation of the Paraloid B72 films and Paraloid B72 treated lime wood samples was evaluated by thermogravimetry throughout the irradiation period of 100 h. The results obtained indicate a shifting of the DTG maxima to lower temperatures, which may be related to a decrease in the stability of the copolymer and wood during photodegradation. The decrease of weight loss with increasing time of exposure was observed, while the global kinetic parameters for the main peak increases when increasing exposure time of wood to the UV light. Even when the surface of the wood was covered with a thin layer of acrylic resin, some photodegradation reactions of the wood surface occurred. The modifications in the wood structure may be influenced by the newly formed structures from acrylic resin photodegradation.     

Myosin-cross-reactive antigen (MCRA) protein from <Emphasis Type="Italic">Bifidobacterium breve</Emphasis> is a FAD-dependent fatty acid hydratase which has a function in stress protection

Background  

The aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates.     

Nano-liquid chromatography-direct electron ionization mass spectrometry: improving performance by a new ion source adapter

A novel interface adapter has been designed to provide a new way of directly coupling a nano-liquid chromatograph to an electron ionization mass spectrometer. It connects the transfer capillary coming from the liquid chromatograph to the ionization chamber and can be easily screwed into the ion source. Liquid coming from the column passes through the heated adapter flow path and is vaporized. A continuous flow of new liquid pushes the vapor into the ionization chamber where it is ionized and continues on to the mass analyzer. The advantages of the new adapter are reduced ice formation inside the ion source and less clogging of the transfer capillary. Improvements achieved are demonstrated on the basis of caffeine and steroid analysis. The limits of detection of selected steroids are compared with and without the adapter. The adapter improves the detection limit of the system by a factor of 2 and precision from ≤15% to ≤9% relative standard deviation. No derivatization procedure is necessary before the analysis of small polar compounds. The resulting spectra are reproducible, easily interpretable, and database searchable. The new method is robust, delivers reproducible results, and provides a highly efficient alternative to existing methods in the field of pharmaceutical analysis.     

The frustrated Lewis pair induced formation of a pentafulvene [6 + 4] cycloaddition product

The frustrated Lewis pair Mes(2)P-CH(2)CH(2)-B(C(6)F(5))(2) reacts with excess 6,6-dimethylpentafulvene to yield a P/B-Lewis pair addition product to an elusive pentafulvene [6 + 4] cycloaddition dimer. This observation may open a new field of utilization of frustrated Lewis pair chemistry.     

New molecular markers for prostate tumor imaging: a study on 2-methylene substituted fatty acids as new AMACR inhibitors

The development of prostate carcinoma is associated with alterations in fatty acid metabolism. α‐Methylacyl‐CoA racemase (AMACR) is a peroxisomal and mitochondrial enzyme that catalyses interconversion between the (S)/(R)‐isomers of a range of α‐methylacyl‐CoA thioesters. AMACR is involved in the β‐oxidation of the dietary branched‐chain fatty acids and bile acid intermediates. It is highly expressed in prostate (more than 95 %), colon (92 %), and breast cancers (44 %) but not in the respective normal or hyperplastic tissues. Thus, targeting of AMACR could be a new strategy for molecular imaging and therapy of prostate and some other cancers. Unlabeled 2‐methylenacyl‐CoA thioesters ( 12 a – c ) were designed as AMACR binding ligands. The thioesters were tested for their ability to inhibit the AMACR‐mediated epimerization of (25R)‐THC‐CoA and were found to be strong AMACR inhibitors. Radioiodinated (E)‐¹³¹I‐13‐iodo‐2‐methylentridec‐12‐enoic acid (¹³¹I‐ 7 c ) demonstrated preferential retention in AMACR‐positive prostate tumor cells (LNCaP, LNCaP C4‐2wt and DU145) compared with both AMACR‐knockout LNCaP C4‐2 AMACR‐siRNA and benign BPH1 prostate cell lines. A significant protein‐bound radioactive fraction with main bands at 47 (sum of molecular weights of AMACR plus 12 c ), 70, and 75 kDa was detected in LNCaP C4‐2 wt cells. In contrast, only negligible amounts of protein‐bound radioactivity were found in LNCaP C4‐2 AMACR‐siRNA cells.