Brevitaxin, a new diterpenolignan from the bark of Taxus brevifolia.

The first terpenolignan, brevitaxin [1], has been isolated from the bark of Taxus brevifolia. Identification was carried out using spectral methods, and the regiochemistry and cytotoxicity of 1 are discussed.

Design and performance of an in-line surface-induced dissociation device in a four-sector mass spectrometer.

A new in-line surface-induced dissociation device has been designed and characterized in a high performance four-sector tandem mass spectrometer. The design incorporates a target electrode parallel to the ion beam axis and an angled deflector plate (45° relative to the ion beam) to provide large collision angles. In addition, an extraction electrode (parallel to the target electrode) is employed to efficiently extract product ions from the target surface. Results obtained with this device indicate high internal energy deposition (up to 16. 3 eV) as measured with the thermometer ions W(CO) 6 (+·) and Si(C2H5)4/+·, as evidenced by extensive dissociation of the refractory pyrene molecular ion, and as indicated by the b 3/y 2 ratio in the product ion spectrum of leucine enkephalin. High resolution provided by the four-sector instrument for both precursor ions and product ions allows the observation of previously unobserved dissociation products in the surface-induced dissociation spectra of Si(C2H5)4/+· and novel ion-surface reaction products in spectra of W(CO)6/+· ions after collisions with hydrocarbon-covered surfaces. Both hydrogen atom and hydrocarbon abstraction products are observed. The dissociation efficiencies measured with the in-line device are approximately 1% when hydrocarbon-coated surfaces are used and increase fivefold with a fluorinated surface.

Human phenol sulfotransferases: chiral substrates and expression in Hep G2 cells.

Enzymatic sulfation of chiral phenolic ethanolamine drugs, e.g. beta-agonists, has been shown to be stereoselective in humans. The reaction appears to be specific for the monoamine (M) form of the phenol sulfotransferases (PSTs). In further studies of the stereochemistry of this reaction, we have found the hepatoblastoma-derived cell line Hep G2 to be an excellent human model. These cells contain the M form PST in quantities exceeding those of human liver by about 4-fold. Thus, sulfate conjugates of the beta-agonist drugs can easily be synthesized for subsequent structural and enzyme kinetic studies. Although less abundant, the phenol (P) form PST as well as dehydroepiandrosterone sulfotransferase are also expressed in the Hep G2 cells.

A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit.

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.

Taxol metabolism in rat hepatocytes.

Metabolism of the anticancer drug taxol was investigated in freshly isolated rat hepatocytes. Two main metabolites were separated by reversed-phase HPLC and shown by tandem mass spectrometry to be monohydroxylated metabolites. Kinetic studies revealed apparent Km values of 68 and 61 microM with identical Vmax values for the two metabolites. Verapamil and midazolam, but not phenacetin, showed concentration-dependent inhibition of taxol metabolism with both metabolites being affected equally. The IC50 was about 100 microM for verapamil and 25 microM for midazolam. These observations demonstrate for the first time in vitro metabolism of taxol and suggest that the metabolism may be subject to potentially important interactions with numerous other drugs.

Stereoselective sulfation of albuterol in humans. Biosynthesis of the sulfate conjugate by HEP G2 cells.

The objective of this study was to test the utility of the human hepatoma cell line Hep G2 for the biosynthesis of the sulfate conjugate of the beta 2-agonist drug albuterol and to determine the stereo-chemistry of the sulfation reaction. Albuterol was, together with Na(2)35SO4, incubated with Hep G2 cells for 48 hr at 37 degrees C. After precipitation of the inorganic sulfate and protein in the medium, the radiolabeled albuterol sulfate formed was isolated by reversed-phase HPLC. The structure of the sulfate conjugate was determined by liquid secondary ion MS and tandem MS, producing a clear molecular ion and a characteristic fragmentation pattern, respectively. The maximum yield from a 100 mm culture dish was 400 micrograms, sufficient for structure identification as well as for development of stereospecific methodology. Efforts to increase the yields by increasing inorganic sulfate or using dexamethasone as an inducer produced only modest effects. Resolution of the albuterol sulfate enantiomers by HPLC, after chiral derivatization, showed a clear preference for the sulfation of (-)-albuterol. This was also shown in kinetic experiments with the individual enantiomers, using cell homogenates, which gave apparent KM values of 115 microM and 528 microM for (-)- and (+)-albuterol, respectively, with an apparent Vmax value 1.7 times higher for (-)-albuterol. The efficiency of sulfation (Vmax/KM) was thus 7.8-fold higher for the pharmacologically more active (-)-enantiomer. Based on these observations, considerably higher bioavailability of (+)- than of (-)-albuterol may be anticipated after oral or inhaled doses of this drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a new fragment ion type in the collision-induced dissociation spectra of peptides: Formation of a2-16 ions.

The identification of an ion observed in the high-energy collision-induced dissociation spectra of several model peptides is reported. The ion, observed at m/z 99 for the peptide pentaalanine (Ala5) and designated a2-16, is shown to have an elemental formula of C5H9NO by high-resolution peak matching. The precursor ion spectrum of the a2-16 ion and product ion spectra of the a2 and the a2+ 1 ions for Ala5 suggest that this ion is formed by the loss of 17 u (presumably NH3) from the a2+1 ion and, to a lesser extent, by loss of 28 u (presumably CO) from the b2-16 ion. On the basis of the data presented and other experimental evidence, a structure and mechanism for the formation of the a2-16 ion is proposed.

Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry.

Bovine rhodopsin has been reported to be S-palmitylated at cysteines 322 and 323 (Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A.S. (1988) FEBS Lett. 230, 1-5). Using a combination of enzymatic and chemical cleavage techniques in conjunction with tandem mass spectrometry, the sites of incorporation of the palmityl groups are shown. Bovine rhodopsin in disc membranes was digested with thermolysin to generate the C-terminal fragment (241-327), which was subsequently cleaved with cyanogen bromide to generate the peptide Val-Thr-Thr-Leu-Cys-Cys-Gly-Lys-Asn-Pro (318-327). A bis-S-palmitylated synthetic standard had the same retention time by reversed-phase high performance liquid chromatography as the isolated peptide and the same molecular weight (MH+1511.7) by liquid secondary ion mass spectrometry. Dithiothreitol reduction of both the isolated and the synthetic peptide cleaved the two thioester-linked palmityl groups to produce reduction products of the same appropriately decreased molecular weight (MH+1035.5). Tandem mass spectrometry of the isolated and the synthetic peptide identified the sites of attachment of the palmityl groups on cysteines 322 and 323. These results prove the modification of cysteines 322 and 323 with palmitic acid in bovine rhodopsin, and illustrate the utility of mass spectrometry to characterize the post-translational modifications in G-protein coupled receptors.

Relations between early childhood care arrangements and college students' psychosocial development and academic performance.

Associations were explored between early substitute care and white middle-class college students' psychosocial development and academic performance. One set of analyses explored associations with the amount of substitute care experienced throughout early childhood (infancy and at ages 2 and 4) and college students' development; another set explored associations between amount of substitute care during infancy (none, part-time, or full-time). Substitute care experience during infancy alone did not differentiate students on the Erikson Psychosocial Inventory Scale (EPSI). There were some near-significant differences on the EPSI among students in different child care arrangements throughout early childhood, but these did not present a consistent pattern. Students' care experience also did not predict the number of extra-curricular activities in which they were presently involved or whether they chose people- or thing-oriented academic majors. However, no day care in infancy followed by full-time day care at ages 2 and 4 was the best predictor of above-average high school academic achievement, and part-time care throughout infancy and early childhood was the best predictor of average high school academic achievement.